Two-colour live-cell nanoscale imaging of intracellular targets

NASA Astrophysics Data System (ADS)

Bottanelli, Francesca; Kromann, Emil B.; Allgeyer, Edward S.; Erdmann, Roman S.; Wood Baguley, Stephanie; Sirinakis, George; Schepartz, Alanna; Baddeley, David; Toomre, Derek K.; Rothman, James E.; Bewersdorf, Joerg

2016-03-01

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution.

Using PSEA-Quant for Protein Set Enrichment Analysis of Quantitative Mass Spectrometry-Based Proteomics.

PubMed

Lavallée-Adam, Mathieu; Yates, John R

2016-03-24

PSEA-Quant analyzes quantitative mass spectrometry-based proteomics datasets to identify enrichments of annotations contained in repositories such as the Gene Ontology and Molecular Signature databases. It allows users to identify the annotations that are significantly enriched for reproducibly quantified high abundance proteins. PSEA-Quant is available on the Web and as a command-line tool. It is compatible with all label-free and isotopic labeling-based quantitative proteomics methods. This protocol describes how to use PSEA-Quant and interpret its output. The importance of each parameter as well as troubleshooting approaches are also discussed. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Sociocultural Theories, Academic Achievement, and African American Adolescents in a Multicultural Context: A Review of the Cultural Compatibility Perspective

ERIC Educational Resources Information Center

Whaley, Arthur L.; Noel, La Tonya

2012-01-01

Several theories suggest that African American culture facilitates academic achievement, but others suggest that identifying with Black culture contributes to the achievement gap by undermining the academic performance among youth. These opposing perspectives are labeled "cultural compatibility theories" and "cultural incompatibility theories,"…

Engineering cell-compatible paper chips for cell culturing, drug screening, and mass spectrometric sensing.

PubMed

Chen, Qiushui; He, Ziyi; Liu, Wu; Lin, Xuexia; Wu, Jing; Li, Haifang; Lin, Jin-Ming

2015-10-28

Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Seven perspectives on GPCR H/D-exchange proteomics methods

PubMed Central

Zhang, Xi

2017-01-01

Recent research shows surging interest to visualize human G protein-coupled receptor (GPCR) dynamic structures using the bottom-up H/D-exchange (HDX) proteomics technology. This opinion article clarifies critical technical nuances and logical thinking behind the GPCR HDX proteomics method, to help scientists overcome cross-discipline pitfalls, and understand and reproduce the protocol at high quality. The 2010 89% HDX structural coverage of GPCR was achieved with both structural and analytical rigor. This article emphasizes systematically considering membrane protein structure stability and compatibility with chromatography and mass spectrometry (MS) throughout the pipeline, including the effects of metal ions, zero-detergent shock, and freeze-thaws on HDX result rigor. This article proposes to view bottom-up HDX as two steps to guide choices of detergent buffers and chromatography settings: (I) protein HDX labeling in native buffers, and (II) peptide-centric analysis of HDX labels, which applies (a) bottom-up MS/MS to construct peptide matrix and (b) HDX MS to locate and quantify H/D labels. The detergent-low-TCEP digestion method demystified the challenge of HDX-grade GPCR digestion. GPCR HDX proteomics is a structural approach, thus its choice of experimental conditions should let structure lead and digestion follow, not the opposite. PMID:28529698

Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection

PubMed Central

Dickerson, Jane A.; Ramsay, Lauren M.; Dada, Oluwatosin O.; Cermak, Nathan

2011-01-01

Capillary isoelectric focusing and capillary zone electrophoresis are coupled with laser-induced fluorescence detection to create an ultrasensitive two-dimensional separation method for proteins. In this method, two capillaries are joined through a buffer filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second dimension separation. A fraction was transferred to the second dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125. PMID:20603830

The quality of social relationships in ravens

PubMed Central

Fraser, Orlaith N.; Bugnyar, Thomas

2015-01-01

The quality of a social relationship represents the history of past social interactions between two individuals, from which the nature and outcome of future interactions can be predicted. Current theory predicts that relationship quality comprises three separate components, its value, compatibility and security. This study is the first to investigate the components of relationship quality in a large-brained bird. Following methods recently used to obtain quantitative measures of each relationship quality component in chimpanzees, Pan troglodytes, we entered data on seven behavioural variables from a group of 11 ravens, Corvus corax, into a principal components analysis. The characteristics of the extracted components matched those predicted for value, compatibility and security, and were labelled as such. When the effects of kinship and sex combination on each relationship quality component were analysed, we found that kin had more valuable relationships, whereas females had less secure and compatible relationships, although the effect of sex combination on compatibility only applied to nonkin. These patterns are consistent with what little knowledge we have of raven relationships from aviary studies and show that the components of relationship quality in ravens may indeed be analogous to those in chimpanzees. PMID:25821236

Multi-stage 3D-2D registration for correction of anatomical deformation in image-guided spine surgery

NASA Astrophysics Data System (ADS)

Ketcha, M. D.; De Silva, T.; Uneri, A.; Jacobson, M. W.; Goerres, J.; Kleinszig, G.; Vogt, S.; Wolinsky, J.-P.; Siewerdsen, J. H.

2017-06-01

A multi-stage image-based 3D-2D registration method is presented that maps annotations in a 3D image (e.g. point labels annotating individual vertebrae in preoperative CT) to an intraoperative radiograph in which the patient has undergone non-rigid anatomical deformation due to changes in patient positioning or due to the intervention itself. The proposed method (termed msLevelCheck) extends a previous rigid registration solution (LevelCheck) to provide an accurate mapping of vertebral labels in the presence of spinal deformation. The method employs a multi-stage series of rigid 3D-2D registrations performed on sets of automatically determined and increasingly localized sub-images, with the final stage achieving a rigid mapping for each label to yield a locally rigid yet globally deformable solution. The method was evaluated first in a phantom study in which a CT image of the spine was acquired followed by a series of 7 mobile radiographs with increasing degree of deformation applied. Second, the method was validated using a clinical data set of patients exhibiting strong spinal deformation during thoracolumbar spine surgery. Registration accuracy was assessed using projection distance error (PDE) and failure rate (PDE  >  20 mm—i.e. label registered outside vertebra). The msLevelCheck method was able to register all vertebrae accurately for all cases of deformation in the phantom study, improving the maximum PDE of the rigid method from 22.4 mm to 3.9 mm. The clinical study demonstrated the feasibility of the approach in real patient data by accurately registering all vertebral labels in each case, eliminating all instances of failure encountered in the conventional rigid method. The multi-stage approach demonstrated accurate mapping of vertebral labels in the presence of strong spinal deformation. The msLevelCheck method maintains other advantageous aspects of the original LevelCheck method (e.g. compatibility with standard clinical workflow, large capture range, and robustness against mismatch in image content) and extends capability to cases exhibiting strong changes in spinal curvature.

Tracking of Engineered Bacteria In Vivo Using Nonstandard Amino Acid Incorporation.

PubMed

Praveschotinunt, Pichet; Dorval Courchesne, Noémie-Manuelle; den Hartog, Ilona; Lu, Chaochen; Kim, Jessica J; Nguyen, Peter Q; Joshi, Neel S

2018-06-15

The rapidly growing field of microbiome research presents a need for better methods of monitoring gut microbes in vivo with high spatial and temporal resolution. We report a method of tracking microbes in vivo within the gastrointestinal tract by programming them to incorporate nonstandard amino acids (NSAA) and labeling them via click chemistry. Using established machinery constituting an orthogonal translation system (OTS), we engineered Escherichia coli to incorporate p-azido-l-phenylalanine (pAzF) in place of the UAG (amber) stop codon. We also introduced a mutant gene encoding for a cell surface protein (CsgA) that was altered to contain an in-frame UAG codon. After pAzF incorporation and extracellular display, the engineered strains could be covalently labeled via copper-free click reaction with a Cy5 dye conjugated to the dibenzocyclooctyl (DBCO) group. We confirmed the functionality of the labeling strategy in vivo using a murine model. Labeling of the engineered strain could be observed using oral administration of the dye to mice several days after colonization of the gastrointestinal tract. This work sets the foundation for the development of in vivo tracking microbial strategies that may be compatible with noninvasive imaging modalities and are capable of longitudinal spatiotemporal monitoring of specific microbial populations.

Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors.

PubMed

Jain, Piyush K; Ramanan, Vyas; Schepers, Arnout G; Dalvie, Nisha S; Panda, Apekshya; Fleming, Heather E; Bhatia, Sangeeta N

2016-09-26

The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. In this work, we have developed a method to photocage the activity of the guide RNA called "CRISPR-plus" (CRISPR-precise light-mediated unveiling of sgRNAs). The photoactivation capability of our CRISPR-plus method is compatible with the simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic investigations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nanoscopy for nanoscience: how super-resolution microscopy extends imaging for nanotechnology.

PubMed

Johnson, Sam A

2015-01-01

Imaging methods have presented scientists with powerful means of investigation for centuries. The ability to resolve structures using light microscopes is though limited to around 200 nm. Fluorescence-based super-resolution light microscopy techniques of several principles and methods have emerged in recent years and offer great potential to extend the capabilities of microscopy. This resolution improvement is especially promising for nanoscience where the imaging of nanoscale structures is inherently restricted by the resolution limit of standard forms of light microscopy. Resolution can be improved by several distinct approaches including structured illumination microscopy, stimulated emission depletion, and single-molecule positioning methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy and several derivative variations of each of these. These methods involve substantial differences in the resolutions achievable in the different axes, speed of acquisition, compatibility with different labels, ease of use, hardware complexity, and compatibility with live biological samples. The field of super-resolution imaging and its application to nanotechnology is relatively new and still rapidly developing. An overview of how these methods may be used with nanomaterials is presented with some examples of pioneering uses of these approaches. © 2014 Wiley Periodicals, Inc.

Configural displays can improve nutrition-related. decisions: an application of the proximity compatibility principle.

PubMed

Marino, Christopher J; Mahan, Robert R

2005-01-01

The nutrition label format currently used by consumers to make dietary-related decisions presents significant information-processing demands for integration-based decisions; however, those demands were not considered as primary factors when the format was adopted. Labels designed in accordance with known principles of cognitive psychology might enhance the kind of decision making that food labeling was intended to facilitate. Three experiments were designed on the basis of the proximity compatibility principle (PCP) to investigate the relationship between nutrition label format and decision making; the experiments involved two types of integration decisions and one type of filtering decision. Based on the PCP, decision performance was measured to test the overall hypothesis that matched task-display tandems would result in better decision performance than would mismatched tandems. In each experiment, a statistically significant increase in mean decision performance was found when the display design was cognitively matched to the demands of the task. Combined, the results from all three experiments support the general hypothesis that task-display matching is a design principle that may enhance the utility of nutrition labeling in nutrition-related decision making. Actual or potential applications of this research include developing robust display solutions that aid in less effortful assimilation of nutrition-related information for consumers.

Direct ultrasensitive electrical detection of prostate cancer biomarkers with CMOS-compatible n- and p-type silicon nanowire sensor arrays.

PubMed

Gao, Anran; Lu, Na; Dai, Pengfei; Fan, Chunhai; Wang, Yuelin; Li, Tie

2014-11-07

Sensitive and quantitative analysis of proteins is central to disease diagnosis, drug screening, and proteomic studies. Here, a label-free, real-time, simultaneous and ultrasensitive prostate-specific antigen (PSA) sensor was developed using CMOS-compatible silicon nanowire field effect transistors (SiNW FET). Highly responsive n- and p-type SiNW arrays were fabricated and integrated on a single chip with a complementary metal oxide semiconductor (CMOS) compatible anisotropic self-stop etching technique which eliminated the need for a hybrid method. The incorporated n- and p-type nanowires revealed complementary electrical response upon PSA binding, providing a unique means of internal control for sensing signal verification. The highly selective, simultaneous and multiplexed detection of PSA marker at attomolar concentrations, a level useful for clinical diagnosis of prostate cancer, was demonstrated. The detection ability was corroborated to be effective by comparing the detection results at different pH values. Furthermore, the real-time measurement was also carried out in a clinically relevant sample of blood serum, indicating the practicable development of rapid, robust, high-performance, and low-cost diagnostic systems.

Extensive Turnover of Compatible Solutes in Cyanobacteria Revealed by Deuterium Oxide (D 2 O) Stable Isotope Probing

DOE PAGES

Baran, Richard; Lau, Rebecca; Bowen, Benjamin P.; ...

2017-01-18

In diverse environments on a global scale cyanobacteria are important primary producers of organic matter. Moreover, while mechanisms of CO 2 fixation are well understood, the distribution of the flow of fixed organic carbon within individual cells and complex microbial communities is less well characterized. To obtain a general overview of metabolism, we describe the use of deuterium oxide (D 2O) to measure deuterium incorporation into the intracellular metabolites of two physiologically diverse cyanobacteria: a terrestrial filamentous strain (Microcoleus vaginatus PCC 9802) and a euryhaline unicellular strain (Synechococcus sp. PCC 7002). D 2O was added to the growth medium duringmore » different phases of the diel cycle. Incorporation of deuterium into metabolites at nonlabile positions, an indicator of metabolite turnover, was assessed using liquid chromatography mass spectrometry. Expectedly, large differences in turnover among metabolites were observed. Some metabolites, such as fatty acids, did not show significant turnover over 12–24 h time periods but did turn over during longer time periods. Unexpectedly, metabolites commonly regarded to act as compatible solutes, including glutamate, glucosylglycerol, and a dihexose, showed extensive turnover compared to most other metabolites already after 12 h, but only during the light phase in the cycle. We observed extensive turnover and found it surprising considering the conventional view on compatible solutes as biosynthetic end points given the relatively slow growth and constant osmotic conditions. Our suggests the possibility of a metabolic sink for some compatible solutes (e.g., into glycogen) that allows for rapid modulation of intracellular osmolarity. To investigate this, uniformly 13C-labeled Synechococcus sp. PCC 7002 were exposed to 12C glucosylglycerol. Following metabolite extraction, amylase treatment of methanol-insoluble polymers revealed 12C labeling of glycogen. Overall, our work shows that D 2O probing is a powerful method for analysis of cyanobacterial metabolism including discovery of novel metabolic processes.« less

Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE™ lightning red.

PubMed

Griebel, Anja; Obermaier, Christian; Westermeier, Reiner; Moche, Martin; Büttner, Knut

2013-07-01

A new fluorescent amino-reactive dye has been tested for both labelling proteins prior to electrophoretic separations and between the two steps of two-dimensional electrophoresis. A series of experiments showed, that the labelling of lysines with this dye is compatible with all standard additives used for sample preparation, including reducing substances and carrier ampholytes. Using this dye for pre-labelling considerably simplifies the electrophoresis and detection workflow and provides highly sensitive and quantitative visualisation of proteins.

Magnetoresistive biosensors for quantitative proteomics

NASA Astrophysics Data System (ADS)

Zhou, Xiahan; Huang, Chih-Cheng; Hall, Drew A.

2017-08-01

Quantitative proteomics, as a developing method for study of proteins and identification of diseases, reveals more comprehensive and accurate information of an organism than traditional genomics. A variety of platforms, such as mass spectrometry, optical sensors, electrochemical sensors, magnetic sensors, etc., have been developed for detecting proteins quantitatively. The sandwich immunoassay is widely used as a labeled detection method due to its high specificity and flexibility allowing multiple different types of labels. While optical sensors use enzyme and fluorophore labels to detect proteins with high sensitivity, they often suffer from high background signal and challenges in miniaturization. Magnetic biosensors, including nuclear magnetic resonance sensors, oscillator-based sensors, Hall-effect sensors, and magnetoresistive sensors, use the specific binding events between magnetic nanoparticles (MNPs) and target proteins to measure the analyte concentration. Compared with other biosensing techniques, magnetic sensors take advantage of the intrinsic lack of magnetic signatures in biological samples to achieve high sensitivity and high specificity, and are compatible with semiconductor-based fabrication process to have low-cost and small-size for point-of-care (POC) applications. Although still in the development stage, magnetic biosensing is a promising technique for in-home testing and portable disease monitoring.

Detection methods and performance criteria for genetically modified organisms.

PubMed

Bertheau, Yves; Diolez, Annick; Kobilinsky, André; Magin, Kimberly

2002-01-01

Detection methods for genetically modified organisms (GMOs) are necessary for many applications, from seed purity assessment to compliance of food labeling in several countries. Numerous analytical methods are currently used or under development to support these needs. The currently used methods are bioassays and protein- and DNA-based detection protocols. To avoid discrepancy of results between such largely different methods and, for instance, the potential resulting legal actions, compatibility of the methods is urgently needed. Performance criteria of methods allow evaluation against a common standard. The more-common performance criteria for detection methods are precision, accuracy, sensitivity, and specificity, which together specifically address other terms used to describe the performance of a method, such as applicability, selectivity, calibration, trueness, precision, recovery, operating range, limit of quantitation, limit of detection, and ruggedness. Performance criteria should provide objective tools to accept or reject specific methods, to validate them, to ensure compatibility between validated methods, and be used on a routine basis to reject data outside an acceptable range of variability. When selecting a method of detection, it is also important to consider its applicability, its field of applications, and its limitations, by including factors such as its ability to detect the target analyte in a given matrix, the duration of the analyses, its cost effectiveness, and the necessary sample sizes for testing. Thus, the current GMO detection methods should be evaluated against a common set of performance criteria.

Cu-Click Compatible Triazabutadienes To Expand the Scope of Aryl Diazonium Ion Chemistry.

PubMed

Cornali, Brandon M; Kimani, Flora W; Jewett, John C

2016-10-07

Triazabutadienes can be used to readily generate reactive aryl diazonium ions under mild, physiologically relevant conditions. These conditions are compatible with a range of functionalities that do not tolerate traditional aryl diazonium ion generation. To increase the utility of this aryl diazonium ion releasing chemistry an alkyne-containing triazabutadiene was synthesized. The copper-catalyzed azide-alkyne cycloaddition ("Cu-click") reaction was utilized to modify the alkyne-containing triazabutadiene and shown to be compatible with the nitrogen-rich triazabutadiene. One of the triazole products was tethered to a fluorophore, thus enabling the direct fluorescent labeling of a model protein.

Digital Equivalent Data System for XRF Labeling of Objects

NASA Technical Reports Server (NTRS)

Schramm, Harry F.; Kaiser, Bruce

2005-01-01

A digital equivalent data system (DEDS) is a system for identifying objects by means of the x-ray fluorescence (XRF) spectra of labeling elements that are encased in or deposited on the objects. As such, a DEDS is a revolutionary new major subsystem of an XRF system. A DEDS embodies the means for converting the spectral data output of an XRF scanner to an ASCII alphanumeric or barcode label that can be used to identify (or verify the assumed or apparent identity of) an XRF-scanned object. A typical XRF spectrum of interest contains peaks at photon energies associated with specific elements on the Periodic Table (see figure). The height of each spectral peak above the local background spectral intensity is proportional to the relative abundance of the corresponding element. Alphanumeric values are assigned to the relative abundances of the elements. Hence, if an object contained labeling elements in suitably chosen proportions, an alphanumeric representation of the object could be extracted from its XRF spectrum. The mixture of labeling elements and for reading the XRF spectrum would be compatible with one of the labeling conventions now used for bar codes and binary matrix patterns (essentially, two-dimensional bar codes that resemble checkerboards). A further benefit of such compatibility is that it would enable the conversion of the XRF spectral output to a bar or matrix-coded label, if needed. In short, a process previously used only for material composition analysis has been reapplied to the world of identification. This new level of verification is now being used for "authentication."

Direct ultrasensitive electrical detection of prostate cancer biomarkers with CMOS-compatible n- and p-type silicon nanowire sensor arrays

NASA Astrophysics Data System (ADS)

Gao, Anran; Lu, Na; Dai, Pengfei; Fan, Chunhai; Wang, Yuelin; Li, Tie

2014-10-01

Sensitive and quantitative analysis of proteins is central to disease diagnosis, drug screening, and proteomic studies. Here, a label-free, real-time, simultaneous and ultrasensitive prostate-specific antigen (PSA) sensor was developed using CMOS-compatible silicon nanowire field effect transistors (SiNW FET). Highly responsive n- and p-type SiNW arrays were fabricated and integrated on a single chip with a complementary metal oxide semiconductor (CMOS) compatible anisotropic self-stop etching technique which eliminated the need for a hybrid method. The incorporated n- and p-type nanowires revealed complementary electrical response upon PSA binding, providing a unique means of internal control for sensing signal verification. The highly selective, simultaneous and multiplexed detection of PSA marker at attomolar concentrations, a level useful for clinical diagnosis of prostate cancer, was demonstrated. The detection ability was corroborated to be effective by comparing the detection results at different pH values. Furthermore, the real-time measurement was also carried out in a clinically relevant sample of blood serum, indicating the practicable development of rapid, robust, high-performance, and low-cost diagnostic systems.Sensitive and quantitative analysis of proteins is central to disease diagnosis, drug screening, and proteomic studies. Here, a label-free, real-time, simultaneous and ultrasensitive prostate-specific antigen (PSA) sensor was developed using CMOS-compatible silicon nanowire field effect transistors (SiNW FET). Highly responsive n- and p-type SiNW arrays were fabricated and integrated on a single chip with a complementary metal oxide semiconductor (CMOS) compatible anisotropic self-stop etching technique which eliminated the need for a hybrid method. The incorporated n- and p-type nanowires revealed complementary electrical response upon PSA binding, providing a unique means of internal control for sensing signal verification. The highly selective, simultaneous and multiplexed detection of PSA marker at attomolar concentrations, a level useful for clinical diagnosis of prostate cancer, was demonstrated. The detection ability was corroborated to be effective by comparing the detection results at different pH values. Furthermore, the real-time measurement was also carried out in a clinically relevant sample of blood serum, indicating the practicable development of rapid, robust, high-performance, and low-cost diagnostic systems. Electronic supplementary information (ESI) available: Electrical characterization of fabricated n- and p-type nanowires, and influence of Debye screening on PSA sensing. See DOI: 10.1039/c4nr03210a

An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP.

PubMed

Bertaccini, Diego; Vaca, Sebastian; Carapito, Christine; Arsène-Ploetze, Florence; Van Dorsselaer, Alain; Schaeffer-Reiss, Christine

2013-06-07

In silico gene prediction has proven to be prone to errors, especially regarding precise localization of start codons that spread in subsequent biological studies. Therefore, the high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and especially proteogenomics fields. The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP) is an efficient N-terminomic approach that allows the characterization of both N-terminal and internal peptides in a single experiment. Due to its permanent positive charge, TMPP labeling strongly affects MS/MS fragmentation resulting in unadapted scoring of TMPP-derivatized peptide spectra by classical search engines. This behavior has led to difficulties in validating TMPP-derivatized peptide identifications with usual score filtering and thus to low/underestimated numbers of identified N-termini. We present herein a new strategy (dN-TOP) that overwhelmed the previous limitation allowing a confident and automated N-terminal peptide validation thanks to a combined labeling with light and heavy TMPP reagents. We show how this double labeling allows increasing the number of validated N-terminal peptides. This strategy represents a considerable improvement to the well-established N-TOP method with an enhanced and accelerated data processing making it now fully compatible with high-throughput proteogenomics studies.

76 FR 44489 - Medical Devices; Neurological Devices; Classification of Repetitive Transcranial Magnetic...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-26

...; Hazards caused by electromagnetic interference and electrostatic discharge hazards; and Hearing loss. FDA... electromagnetic Electromagnetic compatibility. interference and electrostatic discharge hazards. Labeling. Hearing...

Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets.

PubMed

Rant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, Gerhard

2007-10-30

We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 x 10(8) bound targets per cm(2) sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format.

Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets

PubMed Central

Rant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, Gerhard

2007-01-01

We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 × 108 bound targets per cm2 sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format. PMID:17951434

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

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

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

Safety, effectiveness and comparability of professional skin cleansers.

PubMed

Terhaer, Flora K; Bock, Meike; Fartasch, Manigé; Gabard, Bernard; Elsner, Peter; Kleesz, Peter; Landeck, Lilla; Pohrt, Ute; Seyfarth, Florian; Schliemann, Sibylle; Diepgen, Thomas L; Zagrodnik, Fred; John, Swen Malte

2010-10-01

There are no widely-accepted methodical specifications with which to objectify cleansing effectiveness and skin compatibility of occuptional skin cleansing products in Europe. Therefore the German Social Insurance Agency (DGUV) initiated a study with the goal to evaluate such products in view of the potency and the safety of hand cleansers. A market analysis was a part of the project. The product descriptions and safety data sheets of 120 products (5-20/manufacturer) of 11 manufacturers were evaluated between 02/2008 and 04/2008. The manufacturers used mainly ingredients of low irritancy. The declaration of the applied ingredients was in the majority of the cases correctly labeled according to the INCI Declaration. Although there was documentation of skin tolerability for most products, the manufacturers used widely differing tests of skin compatibility. Evidences for cleansing effectiveness were not declared or have not been provided. One manufacturer even promised medical effects of its products. There was no uniform general classification of products making it difficult to identify characteristics of cleansers and choose between them. Presently, there are no commonly accepted criteria to classify products in view of cleansing effectiveness und skin compatibility. Generally accepted criteria and test methods are needed for the evaluation of hand cleansers in order to provide the possibility of transparency and comparability. © The Authors • Journal compilation © Blackwell Verlag GmbH, Berlin.

Learning graph matching.

PubMed

Caetano, Tibério S; McAuley, Julian J; Cheng, Li; Le, Quoc V; Smola, Alex J

2009-06-01

As a fundamental problem in pattern recognition, graph matching has applications in a variety of fields, from computer vision to computational biology. In graph matching, patterns are modeled as graphs and pattern recognition amounts to finding a correspondence between the nodes of different graphs. Many formulations of this problem can be cast in general as a quadratic assignment problem, where a linear term in the objective function encodes node compatibility and a quadratic term encodes edge compatibility. The main research focus in this theme is about designing efficient algorithms for approximately solving the quadratic assignment problem, since it is NP-hard. In this paper we turn our attention to a different question: how to estimate compatibility functions such that the solution of the resulting graph matching problem best matches the expected solution that a human would manually provide. We present a method for learning graph matching: the training examples are pairs of graphs and the 'labels' are matches between them. Our experimental results reveal that learning can substantially improve the performance of standard graph matching algorithms. In particular, we find that simple linear assignment with such a learning scheme outperforms Graduated Assignment with bistochastic normalisation, a state-of-the-art quadratic assignment relaxation algorithm.

DS2 Container and Weatherproofing Study

DTIC Science & Technology

1990-12-01

compatible with polyurethane based coating systems in general? Is it compatible with alkyd -based coatings? 10. How well do the labels placed on the...of the (older) alkyd enamel and (more recently) polyurethane camouflage coatings, leading eventually to seepage of the DS2. A shrink-wrap overpack...by John Wiley and Sons, Inc., Vol. 2, pp. 118- 119; Vol. 4, p. 3 and pp. 284-295; Vol. I, p. 133. 8. Fiber Composite Hybrid Materials, N.L. Hancox

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells.

PubMed

Hofemeier, Arne D; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F W; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-05-26

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO4(3-) symmetric stretch vibrations at 959 cm(-1) assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

NASA Astrophysics Data System (ADS)

Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-05-01

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43- symmetric stretch vibrations at 959 cm-1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

PubMed Central

Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-01-01

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43− symmetric stretch vibrations at 959 cm−1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis. PMID:27225821

Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET.

PubMed

Beeman, Katrin; Baumgärtner, Jens; Laubenheimer, Manuel; Hergesell, Karlheinz; Hoffmann, Martin; Pehl, Ulrich; Fischer, Frank; Pieck, Jan-Carsten

2017-12-01

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated "in-line reader" for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

Agreement of label information of cardiovascular drugs in pregnancy among Korea, the USA, the UK, and Japan.

PubMed

Lee, Shin Haeng; Shin, Ju-Young; Park, Mi-Ju; Park, Byung-Joo

2014-04-01

Drug label is a common source of information; however, the content varies widely. This study aims to evaluate label information on cardiovascular drugs regarding pregnancy for their similarities in Korea, USA, UK, and Japan. Study drugs were selected as following (1) cardiovascular drugs according to the WHO ATC code (C01-C09) and (2) drugs currently marketed in all four countries were included. Evidence level was classified into five categories ('Definite', 'Probable', 'Possible', 'Unlikely', and 'Unclassified') and recommendation level was classified into four categories ('Contraindicated', 'Cautious', 'Compatible', and 'Unclassified'). Frequency and proportion were presented. Percent agreement and kappa coefficient with 95% confidence interval (CI) were calculated using SAS ver. 9.3. Total of 50 cardiovascular drugs were included. 'Unclassified' was represented the most in Korea, followed by Japan and UK (58%, 54%, and 46%, p<0.05). For recommendation level, the majority of drugs in all four countries were classified as 'contraindicated' or 'cautious'. Japanese labels had the largest proportion of 'contraindicated' level (62%), and Korea and UK followed (58%, 44%, p<0.05). Only in the USA, 10.0% of the drugs were 'compatible' whereas, there were none in Korea, UK, and Japan (p<0.01). Korea and Japan showed a substantial agreement in evidence and recommendation level (kappa=0.69, 0.67). Labels of cardiovascular drugs in pregnancy differed widely. Reliable safety information in pregnancy should be provided through regular updates. Copyright © 2014 Elsevier Inc. All rights reserved.

In vivo Labeling of Constellations of Functionally Identified Neurons for Targeted in vitro Recordings

PubMed Central

Lien, Anthony D.; Scanziani, Massimo

2011-01-01

Relating the functional properties of neurons in an intact organism with their cellular and synaptic characteristics is necessary for a mechanistic understanding of brain function. However, while the functional properties of cortical neurons (e.g., tuning to sensory stimuli) are necessarily determined in vivo, detailed cellular and synaptic analysis relies on in vitro techniques. Here we describe an approach that combines in vivo calcium imaging (for functional characterization) with photo-activation of fluorescent proteins (for neuron labeling), thereby allowing targeted in vitro recording of multiple neurons with known functional properties. We expressed photo-activatable GFP rendered non-diffusible through fusion with a histone protein (H2B–PAGFP) in the mouse visual cortex to rapidly photo-label constellations of neurons in vivo at cellular and sub-cellular resolution using two-photon excitation. This photo-labeling method was compatible with two-photon calcium imaging of neuronal responses to visual stimuli, allowing us to label constellations of neurons with specific functional properties. Photo-labeled neurons were easily identified in vitro in acute brain slices and could be targeted for whole-cell recording. We also demonstrate that in vitro and in vivo image stacks of the same photo-labeled neurons could be registered to one another, allowing the exact in vivo response properties of individual neurons recorded in vitro to be known. The ability to perform in vitro recordings from neurons with known functional properties opens up exciting new possibilities for dissecting the cellular, synaptic, and circuit mechanisms that underlie neuronal function in vivo. PMID:22144948

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

PubMed

Hoogenboom, Jorin; Berghuis, Nathalja; Cramer, Dario; Geurts, Rene; Zuilhof, Han; Wennekes, Tom

2016-10-10

Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis.

In-111-labeled leukocytes in the diagnosis of rejection and cytomegalovirus infection in renal transplant patients

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

Forstrom, L.A.; Loken, M.K.; Cook, A.

1981-04-01

Indium-111-labeled (In-111) leukocytes have been shown to be useful in the localization of inflammatory processes, including renal transplant rejection. Using previously reported labeling methods, 63 studies with this agent have been performed in 53 renal transplant patients. Indications for study included suspected rejection or cytomegalovirus (CMV) infection. Studies were performed in 33 men and 20 women, with ages ranging from 6 to 68 years. Autologous cells were normally used for labeling, although leukocytes obtained from ABO-compatible donors were used in three subjects. Rectilinear scanner and/or scintillation camera images were obtained at 24 hours after intravenous administration of 0.1 to 0.6more » mCi of In-111-leukocytes. There was abnormal uptake of In-111-leukocytes in the transplanted kidney in 11 of 15 cases of rejection. In three additional cases of increased transplant uptake, CMV infection was present in two. Abnormal lung uptake was present in 13 of 14 patients with CMV infection. In four additional cases, increased lung uptake was associated with other pulmonary inflammatory disease. Increased lung activity was not seen in patients with uncomplicated transplant rejection. These results suggest that In-111-leukocyte imaging may be useful in the differential diagnosis of rejection versus CMV infection in renal transplant patients.« less

In-111-labeled leukocytes in the diagnosis of rejection and cytomegalovirus infection in renal transplant patients

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

Forstrom, L.A.; Loken, M.K.; Cook, A.

1981-04-01

Indium-111-labelled (In-111) leukocytes have been shown to be useful in the localization of inflammatory processes, including renal transplant rejection. Using previously reported labelling methods, 63 studies with this agent have been performed in 53 renal transplant patients. Indications for study included suspected rejection or cytomegalovirus (CMV) infection. Studies were performed in 33 men and 20 women, with ages ranging from 6 to 68 years. Autologous cells were normally used for labeling, although leukocytes obtained from ABO-compatible donors were used in three subjects. Rectilinear scanner and/or scintillation camera images were obtained at 24 hours after intravenous administration of 0.1 to 0.6more » mCi of In-111 leukocytes. There was abnormal uptake of In-111-leukocytes in the transplanted kidney in 11 of 15 cases of rejection. In three additional cases of increased transplant uptake, CMV infection was present in two. Abnormal lung uptake was present in 13 of 14 patients with CMV infection. In four additional cases, increased lung uptake was associated with other pulmonary inflammatory disease. Increased lung activity was not seen in patients with uncomplicated transplant rejection. These results suggest that In-111-leukocyte imaging may be useful in the differential diagnosis of rejection versus CMV infection in renal transplant patients.« less

Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

PubMed Central

Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P.; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

2016-01-01

The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

Fe(OTf)3-catalysed Friedel–Crafts reaction of benzenoid arenes with α,β-unsaturated carbonyl compounds: easy access to 1,1-diarylalkanes

PubMed Central

Bhattacharya, Aditya; Shukla, Pushpendra Mani

2017-01-01

A simple and efficient method for the synthesis of 1,1-diarylalkanes via the Friedel–Crafts-type alkylation reaction of electron-rich arenes with cinnamic acid ester derivatives or chalcones is reported. Iron triflate has been found to be the best catalyst for the Friedel–Crafts-type alkylation reaction with α,β-unsaturated carbonyl compounds. This reaction afforded β,β-diaryl carbonyl compounds in good yields (65–93%) and with excellent regioselectivities. Remarkably, this method is also compatible with a variety of indoles to provide 3-indolyl-aryl carbonyl compounds in excellent yields. Great efforts have been made to deduce a plausible reaction mechanism based on isotopic labelling experiments. PMID:29134078

Development of isotope labeling LC-MS for human salivary metabolomics and application to profiling metabolome changes associated with mild cognitive impairment.

PubMed

Zheng, Jiamin; Dixon, Roger A; Li, Liang

2012-12-18

Saliva is a readily available biofluid that may contain metabolites of interest for diagnosis and prognosis of diseases. In this work, a differential (13)C/(12)C isotope dansylation labeling method, combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), is described for quantitative profiling of the human salivary metabolome. New strategies are presented to optimize the sample preparation and LC-MS detection processes. The strategies allow the use of as little of 5 μL of saliva sample as a starting material to determine the concentration changes of an average of 1058 ion pairs or putative metabolites in comparative saliva samples. The overall workflow consists of several steps including acetone-induced protein precipitation, (12)C-dansylation labeling of the metabolites, and LC-UV measurement of the total concentration of the labeled metabolites in individual saliva samples. A pooled sample was prepared from all the individual samples and labeled with (13)C-dansylation to serve as a reference. Using this metabolome profiling method, it was found that compatible metabolome results could be obtained after saliva samples were stored in tubes normally used for genetic material collection at room temperature, -20 °C freezer, and -80 °C freezer over a period of 1 month, suggesting that many saliva samples already collected in genomic studies could become a valuable resource for metabolomics studies, although the effect of much longer term of storage remains to be determined. Finally, the developed method was applied for analyzing the metabolome changes of two different groups: normal healthy older adults and comparable older adults with mild cognitive impairment (MCI). Top-ranked 18 metabolites successfully distinguished the two groups, among which seven metabolites were putatively identified while one metabolite, taurine, was definitively identified.

21 CFR 874.3305 - Wireless air-conduction hearing aid.

Code of Federal Regulations, 2013 CFR

2013-04-01

...-amplifying device, intended to compensate for impaired hearing that incorporates wireless technology in its...: (1) Appropriate analysis/testing should validate electro magnetic compatibility (EMC) and safety of... technology functions; and (3) Labeling should specify appropriate instructions, warnings, and information...

Microplate-compatible total internal reflection fluorescence microscopy for receptor pharmacology

NASA Astrophysics Data System (ADS)

Chen, Minghan; Zaytseva, Natalya V.; Wu, Qi; Li, Min; Fang, Ye

2013-05-01

We report the use of total internal reflection fluorescence (TIRF) microscopy for analyzing receptor pharmacology and the development of a microplate-compatible TIRF imaging system. Using stably expressed green fluorescence protein tagged β2-adrenergic receptor as the reporter, we found that the activation of different receptors results in distinct kinetic signatures of the TIRF intensity of cells. These TIRF signatures closely resemble the characteristics of their respective label-free dynamic mass redistribution signals in the same cells. This suggests that TIRF in microplate can be used for profiling and screening drugs.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei.

PubMed

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

2016-07-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes) or TINA-DNA (Twisted Intercalating Nucleic Acids). Gene targets can be specifically labelled with at least about 20 probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3d-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. Copyright © 2016. Published by Elsevier Inc.

Embodiment of abstract categories in space… grounding or mere compatibility effects? The case of politics.

PubMed

Farias, Ana Rita; Garrido, Margarida V; Semin, Gün R

2016-05-01

In two experiments, the role played by stimulus response compatibility in driving the spatial grounding of abstract concepts is examined. In Experiment 1, participants were asked to classify politics-related words appearing to the left or the right side of a computer monitor as socialist or conservative. Responses were given by pressing vertically aligned keys and thus orthogonal to the spatial information that may have been implied by the words. Responses given by left or right index finger were counterbalanced. In Experiment 2, a lexical decision task, participants categorized political words or non-words presented to the left or the right auditory channels, by pressing the top/bottom button of a response box. The response category labels (word or non-word) were also orthogonal to the spatial information that may have been implied by the stimulus words. In both experiments, responses were faster when socialism-related words were presented on the left and conservatism-related words were presented on the right, irrespective of the reference of the response keys or labels. Overall, our findings suggest that the spatial grounding of abstract concepts (or at least politics-related ones) is independent of experimentally driven stimulus-response compatibility effects. Copyright © 2016 Elsevier B.V. All rights reserved.

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid.

PubMed

Anumula, K R; Dhume, S T

1998-07-01

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2-aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.

Size-matched alkyne-conjugated cyanine fluorophores to identify differences in protein glycosylation.

PubMed

Burnham-Marusich, Amanda R; Plechaty, Anna M; Berninsone, Patricia M

2014-09-01

Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D-DIGE. In "Click-DIGE", posttranslationally modified proteins are metabolically labeled with azido-substrate analogs, then size- and charge-matched alkyne-Cy3 or alkyne-Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently-tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click-DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click-DIGE specifically labels azido proteins, (ii) the resulting Cy-protein conjugates are spectrally distinct, and (iii) the conjugates are size- and charge-matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP-galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click-DIGE to be compatible with analysis of a wide range of PTMs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Eat Me If You Can: Cognitive Mechanisms Underlying the Distance Effect

PubMed Central

Junghans, Astrid F.; Evers, Catharine; De Ridder, Denise T. D.

2013-01-01

Proximal objects provide affordances that activate the motor information involved in interacting with the objects. This effect has previously been shown for artifacts but not for natural objects, such as food. This study examined whether the sight of proximal food, compared to distant food activates eating-related information. In two experiments reaction times to verbal labels following the sight of proximal and distant objects (food and toys) were measured. Verbal labels included function words that were compatible with one object category (eating and playing) and observation words compatible with both object categories. The sight of food was expected to activate eating-related information when presented at proximity but not at distance, as reflected by faster reaction times to proximal than distant compatible eating words and no difference between reaction times to proximal and distant food for observation words (Experiment 1). Experiment 2 additionally compared the reaction times to wrapped and unwrapped food. The distance effect was expected to occur only for unwrapped food because only unwrapped food is readily edible. As expected, Experiment 1 and 2 revealed faster responses to compatible eating words at proximity than at distance. In Experiment 2 this distance effect occurred only for readily edible, unwrapped food but not for wrapped food. For observation words no difference in response times between the distances was found. These findings suggest that the sight of proximal food activates eating-related information, which could explain people’s differential behavioral responses to reachable versus distant food. The activation of eating-related information upon sight of accessible food could provide a cognition-based explanation for mindless eating. PMID:24367684

3D multiplexed immunoplasmonics microscopy

NASA Astrophysics Data System (ADS)

Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

2016-07-01

Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence.Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence. Electronic supplementary information (ESI) available: Characterization of functionalized nanoparticles by UV-visible-NIR spectroscopy, standard dark field microscopy and reflected light microscopy. Immunofluorescence of cells. See DOI: 10.1039/c6nr01257d

Converting HAZUS capacity curves to seismic hazard-compatible building fragility functions: effect of hysteretic models

USGS Publications Warehouse

Ryu, Hyeuk; Luco, Nicolas; Baker, Jack W.; Karaca, Erdem

2008-01-01

A methodology was recently proposed for the development of hazard-compatible building fragility models using parameters of capacity curves and damage state thresholds from HAZUS (Karaca and Luco, 2008). In the methodology, HAZUS curvilinear capacity curves were used to define nonlinear dynamic SDOF models that were subjected to the nonlinear time history analysis instead of the capacity spectrum method. In this study, we construct a multilinear capacity curve with negative stiffness after an ultimate (capping) point for the nonlinear time history analysis, as an alternative to the curvilinear model provided in HAZUS. As an illustration, here we propose parameter values of the multilinear capacity curve for a moderate-code low-rise steel moment resisting frame building (labeled S1L in HAZUS). To determine the final parameter values, we perform nonlinear time history analyses of SDOF systems with various parameter values and investigate their effects on resulting fragility functions through sensitivity analysis. The findings improve capacity curves and thereby fragility and/or vulnerability models for generic types of structures.

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

PubMed Central

Qi, Xiaoquan; Bakht, Saleha; Devos, Katrien M.; Gale, Mike D.; Osbourn, Anne

2001-01-01

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays. PMID:11713336

High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts

PubMed Central

Hoffmann, Stefanie; Walter, Steffi; Blume, Anne-Kathrin; Fuchs, Stephan; Schmidt, Christiane; Scholz, Annemarie; Gerlach, Roman G.

2018-01-01

The quantification of bacteria in cell culture infection models is of paramount importance for the characterization of host-pathogen interactions and pathogenicity factors involved. The standard to enumerate bacteria in these assays is plating of a dilution series on solid agar and counting of the resulting colony forming units (CFU). In contrast, the virtual colony count (VCC) method is a high-throughput compatible alternative with minimized manual input. Based on the recording of quantitative growth kinetics, VCC relates the time to reach a given absorbance threshold to the initial cell count using a series of calibration curves. Here, we adapted the VCC method using the model organism Salmonella enterica sv. Typhimurium (S. Typhimurium) in combination with established cell culture-based infection models. For HeLa infections, a direct side-by-side comparison showed a good correlation of VCC with CFU counting after plating. For MDCK cells and RAW macrophages we found that VCC reproduced the expected phenotypes of different S. Typhimurium mutants. Furthermore, we demonstrated the use of VCC to test the inhibition of Salmonella invasion by the probiotic E. coli strain Nissle 1917. Taken together, VCC provides a flexible, label-free, automation-compatible methodology to quantify bacteria in in vitro infection assays. PMID:29497603

Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

PubMed Central

Xiong, Hanqing; Zhou, Zhenqiao; Zhu, Mingqiang; Lv, Xiaohua; Li, Anan; Li, Shiwei; Li, Longhui; Yang, Tao; Wang, Siming; Yang, Zhongqin; Xu, Tonghui; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2014-01-01

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding. PMID:24886825

A METHOD FOR DETERMINING THE COMPATIBILITY OF HAZARDOUS WASTES

EPA Science Inventory

This report describes a method for determining the compatibility of the binary combinations of hazardous wastes. The method consists of two main parts, namely: (1) the step-by-step compatibility analysis procedures, and (2) the hazardous wastes compatibility chart. The key elemen...

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

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

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with atmore » least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.« less

Interfacial polymerization for colorimetric labeling of protein expression in cells.

PubMed

Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

2014-01-01

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

Bacterial Production of Site Specific 13C Labeled Phenylalanine and Methodology for High Level Incorporation into Bacterially Expressed Recombinant Proteins

PubMed Central

Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L.

2016-01-01

Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study. PMID:28028744

Optical diffraction tomography with fully and partially coherent illumination in high numerical aperture label-free microscopy [Invited].

PubMed

Soto, Juan M; Rodrigo, José A; Alieva, Tatiana

2018-01-01

Quantitative label-free imaging is an important tool for the study of living microorganisms that, during the last decade, has attracted wide attention from the optical community. Optical diffraction tomography (ODT) is probably the most relevant technique for quantitative label-free 3D imaging applied in wide-field microscopy in the visible range. The ODT is usually performed using spatially coherent light illumination and specially designed holographic microscopes. Nevertheless, the ODT is also compatible with partially coherent illumination and can be realized in conventional wide-field microscopes by applying refocusing techniques, as it has been recently demonstrated. Here, we compare these two ODT modalities, underlining their pros and cons and discussing the optical setups for their implementation. In particular, we pay special attention to a system that is compatible with a conventional wide-field microscope that can be used for both ODT modalities. It consists of two easily attachable modules: the first for sample illumination engineering based on digital light processing technology; the other for focus scanning by using an electrically driven tunable lens. This hardware allows for a programmable selection of the wavelength and the illumination design, and provides fast data acquisition as well. Its performance is experimentally demonstrated in the case of ODT with partially coherent illumination providing speckle-free 3D quantitative imaging.

Integrated polymer-based Mach-Zehnder interferometer label-free streptavidin biosensor compatible with injection molding.

PubMed

Bruck, R; Melnik, E; Muellner, P; Hainberger, R; Lämmerhofer, M

2011-05-15

We report the development of a Mach-Zehnder interferometer biosensor based on a high index contrast polymer material system and the demonstration of label-free online measurement of biotin-streptavidin binding on the sensor surface. The surface of the polyimide waveguide core layer was functionalized with 3-mercaptopropyl trimethoxy silane and malemide tagged biotin. Several concentrations of Chromeon 642-streptavidin dissolved in phosphate buffered saline solution were rinsed over the functionalized sensor surface by means of a fluidic system and the biotin-streptavidin binding process was observed in the output signal of the interferometer at a wavelength of 1310 nm. Despite the large wavelength and the comparatively low surface sensitivity of the sensor system due to the low index contrast in polymer material systems compared to inorganic material systems, we were able to resolve streptavidin concentrations of down to 0.1 μg/ml. The polymer-based optical sensor design is fully compatible with cost-efficient mass production technologies such as injection molding and spin coating, which makes it an attractive alternative to inorganic optical sensors. Copyright © 2011 Elsevier B.V. All rights reserved.

Monitoring Cell Proliferation by Dye Dilution: Considerations for Probe Selection

PubMed Central

Tario, Joseph D.; Conway, Alexis N.; Muirhead, Katharine A.; Wallace, Paul K.

2018-01-01

In the third edition of this series, we described protocols for labeling cell populations with tracking dyes, and addressed issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T cell functions. We summarized key characteristics of and differences between general protein and membrane labeling dyes, discussed determination of optimal staining concentrations, and provided detailed labeling protocols for both dye types. Examples of the advantages of two color cell tracking were provided in the form of protocols for: (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay; and (b) an in vitro suppression assay for simultaneous proliferation monitoring of effector and regulatory T cells. The number of commercially available fluorescent cell tracking dyes has expanded significantly since the last edition, with new suppliers and/or new spectral properties being added at least annually. In this fourth edition, we describe evaluations to be performed by the supplier and/or user when characterizing a new cell tracking dye and by the user when selecting one for use in multicolor proliferation monitoring. These include methods for: Assessment of the dye’s spectral profile on the laboratory’s flow cytometer(s) to optimize compatibility with other employed fluorochromes and minimize compensation problems;Evaluating the effect of labeling on cell growth rate;Testing the fidelity with which dye dilution reports cell division;Determining the maximum number of generations to be included when using dye dilution profiles to estimate fold population expansion or frequency of responder cells; andVerifying that relevant cell functions (e.g., effector activity) remain unaltered by tracking dye labeling. PMID:29071683

Efficient FPT Algorithms for (Strict) Compatibility of Unrooted Phylogenetic Trees.

PubMed

Baste, Julien; Paul, Christophe; Sau, Ignasi; Scornavacca, Celine

2017-04-01

In phylogenetics, a central problem is to infer the evolutionary relationships between a set of species X; these relationships are often depicted via a phylogenetic tree-a tree having its leaves labeled bijectively by elements of X and without degree-2 nodes-called the "species tree." One common approach for reconstructing a species tree consists in first constructing several phylogenetic trees from primary data (e.g., DNA sequences originating from some species in X), and then constructing a single phylogenetic tree maximizing the "concordance" with the input trees. The obtained tree is our estimation of the species tree and, when the input trees are defined on overlapping-but not identical-sets of labels, is called "supertree." In this paper, we focus on two problems that are central when combining phylogenetic trees into a supertree: the compatibility and the strict compatibility problems for unrooted phylogenetic trees. These problems are strongly related, respectively, to the notions of "containing as a minor" and "containing as a topological minor" in the graph community. Both problems are known to be fixed parameter tractable in the number of input trees k, by using their expressibility in monadic second-order logic and a reduction to graphs of bounded treewidth. Motivated by the fact that the dependency on k of these algorithms is prohibitively large, we give the first explicit dynamic programming algorithms for solving these problems, both running in time [Formula: see text], where n is the total size of the input.

Methods for fabrication of flexible hybrid electronics

NASA Astrophysics Data System (ADS)

Street, Robert A.; Mei, Ping; Krusor, Brent; Ready, Steve E.; Zhang, Yong; Schwartz, David E.; Pierre, Adrien; Doris, Sean E.; Russo, Beverly; Kor, Siv; Veres, Janos

2017-08-01

Printed and flexible hybrid electronics is an emerging technology with potential applications in smart labels, wearable electronics, soft robotics, and prosthetics. Printed solution-based materials are compatible with plastic film substrates that are flexible, soft, and stretchable, thus enabling conformal integration with non-planar objects. In addition, manufacturing by printing is scalable to large areas and is amenable to low-cost sheet-fed and roll-to-roll processes. FHE includes display and sensory components to interface with users and environments. On the system level, devices also require electronic circuits for power, memory, signal conditioning, and communications. Those electronic components can be integrated onto a flexible substrate by either assembly or printing. PARC has developed systems and processes for realizing both approaches. This talk presents fabrication methods with an emphasis on techniques recently developed for the assembly of off-the-shelf chips. A few examples of systems fabricated with this approach are also described.

Raman Plus X: Biomedical Applications of Multimodal Raman Spectroscopy.

PubMed

Das, Nandan K; Dai, Yichuan; Liu, Peng; Hu, Chuanzhen; Tong, Lieshu; Chen, Xiaoya; Smith, Zachary J

2017-07-07

Raman spectroscopy is a label-free method of obtaining detailed chemical information about samples. Its compatibility with living tissue makes it an attractive choice for biomedical analysis, yet its translation from a research tool to a clinical tool has been slow, hampered by fundamental Raman scattering issues such as long integration times and limited penetration depth. In this review we detail the how combining Raman spectroscopy with other techniques yields multimodal instruments that can help to surmount the translational barriers faced by Raman alone. We review Raman combined with several optical and non-optical methods, including fluorescence, elastic scattering, OCT, phase imaging, and mass spectrometry. In each section we highlight the power of each combination along with a brief history and presentation of representative results. Finally, we conclude with a perspective detailing both benefits and challenges for multimodal Raman measurements, and give thoughts on future directions in the field.

Raman Plus X: Biomedical Applications of Multimodal Raman Spectroscopy

PubMed Central

Das, Nandan K.; Dai, Yichuan; Liu, Peng; Hu, Chuanzhen; Tong, Lieshu; Chen, Xiaoya

2017-01-01

Raman spectroscopy is a label-free method of obtaining detailed chemical information about samples. Its compatibility with living tissue makes it an attractive choice for biomedical analysis, yet its translation from a research tool to a clinical tool has been slow, hampered by fundamental Raman scattering issues such as long integration times and limited penetration depth. In this review we detail the how combining Raman spectroscopy with other techniques yields multimodal instruments that can help to surmount the translational barriers faced by Raman alone. We review Raman combined with several optical and non-optical methods, including fluorescence, elastic scattering, OCT, phase imaging, and mass spectrometry. In each section we highlight the power of each combination along with a brief history and presentation of representative results. Finally, we conclude with a perspective detailing both benefits and challenges for multimodal Raman measurements, and give thoughts on future directions in the field. PMID:28686212

47 CFR 61.22 - Composition of tariffs.

Code of Federal Regulations, 2011 CFR

2011-10-01

.../2 inch (8.89 cm) diskette, or a 5 inch CD-ROM, formatted in an IBM-compatible form using either Word... tariff. The diskette or CD-ROM must be submitted in “read only” mode. The diskette or CD-ROM must be... multiple diskettes or CD-ROMs are submitted, the issuing carrier shall clearly label each diskette in the...

78 FR 9349 - Medical Devices; Ophthalmic Devices; Classification of the Eyelid Weight

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-08

... MR incompatibility or the conditions for safe use in an MR environment. Labeling will mitigate the... Amendments of 1976 (Pub. L. 94-295), the Safe Medical Devices Act of 1990 (Pub. L. 101-629), and the Food and...) and nonclinical testing evaluating the compatibility of the device in a MR environment. In addition...

Site-Specific Three-Color Labeling of α-Synuclein via Conjugation to Uniquely Reactive Cysteines during Assembly by Native Chemical Ligation.

PubMed

Lee, Taehyung C; Moran, Crystal R; Cistrone, Philip A; Dawson, Philip E; Deniz, Ashok A

2018-04-12

Single-molecule fluorescence is widely used to study conformational complexity in proteins, and has proven especially valuable with intrinsically disordered proteins (IDPs). Protein studies using dual-color single-molecule Förster resonance energy transfer (smFRET) are now quite common, but many could benefit from simultaneous measurement of multiple distances through multi-color labeling. Such studies, however, have suffered from limitations in site-specific incorporation of more than two dyes per polypeptide. Here we present a fully site-specific three-color labeling scheme for α-synuclein, an IDP with important putative functions and links to Parkinson disease. The convergent synthesis combines native chemical ligation with regiospecific cysteine protection of expressed protein fragments to permit highly controlled labeling via standard cysteine-maleimide chemistry, enabling more global smFRET studies. Furthermore, this modular approach is generally compatible with recombinant proteins and expandable to accommodate even more complex experiments, such as by labeling with additional colors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Neuro-Compatible Metabolic Glycan Labeling of Primary Hippocampal Neurons in Noncontact, Sandwich-Type Neuron-Astrocyte Coculture.

PubMed

Choi, Ji Yu; Park, Matthew; Cho, Hyeoncheol; Kim, Mi-Hee; Kang, Kyungtae; Choi, Insung S

2017-12-20

Glycans are intimately involved in several facets of neuronal development and neuropathology. However, the metabolic labeling of surface glycans in primary neurons is a difficult task because of the neurotoxicity of unnatural monosaccharides that are used as a metabolic precursor, hindering the progress of metabolic engineering in neuron-related fields. Therefore, in this paper, we report a neurosupportive, neuron-astrocyte coculture system that neutralizes the neurotoxic effects of unnatural monosaccharides, allowing for the long-term observation and characterization of glycans in primary neurons in vitro. Polysialic acids in neurons are selectively imaged, via the metabolic labeling of sialoglycans with peracetylated N-azidoacetyl-d-mannosamine (Ac 4 ManNAz), for up to 21 DIV. Two-color labeling shows that neuronal activities, such as neurite outgrowth and recycling of membrane components, are highly dynamic and change over time during development. In addition, the insertion sites of membrane components are suggested to not be random, but be predominantly localized in developing neurites. This work provides a new research platform and also suggests advanced 3D systems for metabolic-labeling studies of glycans in primary neurons.

Compatibility Conditions of Structural Mechanics

NASA Technical Reports Server (NTRS)

Patnaik, Surya N.; Coroneos, Rula M.; Hopkins, Dale A.

1999-01-01

The theory of elasticity has camouflaged a deficiency in the compatibility formulation since 1860. In structures the ad hoc compatibility conditions through virtual "cuts" and closing "gaps" are not parallel to the strain formulation in elasticity. This deficiency in the compatibility conditions has prevented the development of a direct stress determination method in structures and in elasticity. We have addressed this deficiency and attempted to unify the theory of compatibility. This work has led to the development of the integrated force method for structures and the completed Beltrami-Michell formulation for elasticity. The improved accuracy observed in the solution of numerical examples by the integrated force method can be attributed to the compliance of the compatibility conditions. Using the compatibility conditions allows mapping of variables and facile movement among different structural analysis formulations. This paper reviews and illustrates the requirement of compatibility in structures and in elasticity. It also describes the generation of the conditions and quantifies the benefits of their use. The traditional analysis methods and available solutions (which have been obtained bypassing the missed conditions) should be verified for compliance of the compatibility conditions.

Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

PubMed Central

Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J.

2014-01-01

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium. PMID:25536421

Single-neuron labeling with inducible cre-mediated knockout in transgenic mice

PubMed Central

Young, Paul; Qiu, Li; Wang, Dongqing; Zhao, Shengli; Gross, James; Feng, Guoping

2011-01-01

To facilitate functional analysis of neuronal connectivity in a mammalian nervous system tightly packed with billions of cells, we developed a new technique that allows inducible genetic manipulations within fluorescently labeled single neurons in mice. We term this technique SLICK for Single-neuron Labeling with Inducible Cre-mediated Knockout. SLICK is achieved by co-expressing a drug-inducible form of cre recombinase and a fluorescent protein within the same small subsets of neurons. Thus, SLICK combines the powerful cre recombinase system for conditional genetic manipulation and the fluorescent labeling of single neurons for imaging. We demonstrate efficient inducible genetic manipulation in several types of neurons using SLICK. Furthermore, we apply SLICK to eliminate synaptic transmission in a small subset of neuromuscular junctions. Our results provide evidence for the long-term stability of inactive neuromuscular synapses in adult animals. More broadly, these studies demonstrate a cre-LoxP compatible system for dissecting gene functions in single identifiable neurons. PMID:18454144

A sensitive analytical procedure for monitoring acrylamide in environmental water samples by offline SPE-UPLC/MS/MS.

PubMed

Togola, Anne; Coureau, Charlotte; Guezennec, Anne-Gwenaëlle; Touzé, Solène

2015-05-01

The presence of acrylamide in natural systems is of concern from both environmental and health points of view. We developed an accurate and robust analytical procedure (offline solid phase extraction combined with UPLC/MS/MS) with a limit of quantification (20 ng L(-1)) compatible with toxicity threshold values. The optimized (considering the nature of extraction phases, sampling volumes, and solvent of elution) solid phase extraction (SPE) was validated according to ISO Standard ISO/IEC 17025 on groundwater, surface water, and industrial process water samples. Acrylamide is highly polar, which induces a high variability during the SPE step, therefore requiring the use of C(13)-labeled acrylamide as an internal standard to guarantee the accuracy and robustness of the method (uncertainty about 25 % (k = 2) at limit of quantification level). The specificity of the method and the stability of acrylamide were studied for these environmental media, and it was shown that the method is suitable for measuring acrylamide in environmental studies.

Segmentation of Image Ensembles via Latent Atlases

PubMed Central

Van Leemput, Koen; Menze, Bjoern H.; Wells, William M.; Golland, Polina

2010-01-01

Spatial priors, such as probabilistic atlases, play an important role in MRI segmentation. However, the availability of comprehensive, reliable and suitable manual segmentations for atlas construction is limited. We therefore propose a method for joint segmentation of corresponding regions of interest in a collection of aligned images that does not require labeled training data. Instead, a latent atlas, initialized by at most a single manual segmentation, is inferred from the evolving segmentations of the ensemble. The algorithm is based on probabilistic principles but is solved using partial differential equations (PDEs) and energy minimization criteria. We evaluate the method on two datasets, segmenting subcortical and cortical structures in a multi-subject study and extracting brain tumors in a single-subject multi-modal longitudinal experiment. We compare the segmentation results to manual segmentations, when those exist, and to the results of a state-of-the-art atlas-based segmentation method. The quality of the results supports the latent atlas as a promising alternative when existing atlases are not compatible with the images to be segmented. PMID:20580305

Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

PubMed

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

2014-11-04

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

Application of Adenosine Triphosphate Affinity Probe and Scheduled Multiple-Reaction Monitoring Analysis for Profiling Global Kinome in Human Cells in Response to Arsenite Treatment

PubMed Central

2015-01-01

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli. PMID:25301106

Silica-coated quantum dots for optical evaluation of perfluorocarbon droplet interactions with cells.

PubMed

Gorelikov, Ivan; Martin, Amanda L; Seo, Minseok; Matsuura, Naomi

2011-12-20

There has been recent interest in developing new, targeted, perfluorocarbon (PFC) droplet-based contrast agents for medical imaging (e.g., magnetic resonance imaging, X-ray/computed tomography, and ultrasound imaging). However, due to the large number of potential PFCs and droplet stabilization strategies available, it is challenging to determine in advance the PFC droplet formulation that will result in the optimal in vivo behavior and imaging performance required for clinical success. We propose that the integration of fluorescent quantum dots (QDs) into new PFC droplet agents can help to rapidly screen new PFC-based candidate agents for biological compatibility early in their development. QD labels can allow the interaction of PFC droplets with single cells to be assessed at high sensitivity and resolution using optical methods in vitro, complementing the deeper depth penetration but lower resolution provided by PFC droplet imaging using in vivo medical imaging systems. In this work, we introduce a simple and robust method to miscibilize silica-coated nanoparticles into hydrophobic and lipophobic PFCs through fluorination of the silica surface via a hydrolysis-condensation reaction with 1H,1H,2H,2H-perfluorodecyltriethoxysilane. Using CdSe/ZnS core/shell QDs, we show that nanoscale, QD-labeled PFC droplets can be easily formed, with similar sizes and surface charges as unlabeled PFC droplets. The QD label can be used to determine the PFC droplet uptake into cells in vitro by fluorescence microscopy and flow cytometry, and can be used to validate the fate of PFC droplets in vivo in small animals via fluorescence microscopy of histological tissue sections. This is demonstrated in macrophage and cancer cells, and in rabbits, respectively. This work reveals the potential of using QD labels for rapid, preclinical, optical assessment of different PFC droplet formulations for their future use in patients. © 2011 American Chemical Society

Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

PubMed

Paiva, Ana L S; Oliveira, Jose T A; de Souza, Gustavo A; Vasconcelos, Ilka M

2016-12-02

Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

Droplet-based Biosensing for Lab-on-a-Chip, Open Microfluidics Platforms

PubMed Central

Dak, Piyush; Ebrahimi, Aida; Swaminathan, Vikhram; Duarte-Guevara, Carlos; Bashir, Rashid; Alam, Muhammad A.

2016-01-01

Low cost, portable sensors can transform health care by bringing easily available diagnostic devices to low and middle income population, particularly in developing countries. Sample preparation, analyte handling and labeling are primary cost concerns for traditional lab-based diagnostic systems. Lab-on-a-chip (LoC) platforms based on droplet-based microfluidics promise to integrate and automate these complex and expensive laboratory procedures onto a single chip; the cost will be further reduced if label-free biosensors could be integrated onto the LoC platforms. Here, we review some recent developments of label-free, droplet-based biosensors, compatible with “open” digital microfluidic systems. These low-cost droplet-based biosensors overcome some of the fundamental limitations of the classical sensors, enabling timely diagnosis. We identify the key challenges that must be addressed to make these sensors commercially viable and summarize a number of promising research directions. PMID:27089377

Droplet-based Biosensing for Lab-on-a-Chip, Open Microfluidics Platforms.

PubMed

Dak, Piyush; Ebrahimi, Aida; Swaminathan, Vikhram; Duarte-Guevara, Carlos; Bashir, Rashid; Alam, Muhammad A

2016-04-14

Low cost, portable sensors can transform health care by bringing easily available diagnostic devices to low and middle income population, particularly in developing countries. Sample preparation, analyte handling and labeling are primary cost concerns for traditional lab-based diagnostic systems. Lab-on-a-chip (LoC) platforms based on droplet-based microfluidics promise to integrate and automate these complex and expensive laboratory procedures onto a single chip; the cost will be further reduced if label-free biosensors could be integrated onto the LoC platforms. Here, we review some recent developments of label-free, droplet-based biosensors, compatible with "open" digital microfluidic systems. These low-cost droplet-based biosensors overcome some of the fundamental limitations of the classical sensors, enabling timely diagnosis. We identify the key challenges that must be addressed to make these sensors commercially viable and summarize a number of promising research directions.

Label-Free Bioanalyte Detection from Nanometer to Micrometer Dimensions-Molecular Imprinting and QCMs †.

PubMed

Mujahid, Adnan; Mustafa, Ghulam; Dickert, Franz L

2018-06-01

Modern diagnostic tools and immunoassay protocols urges direct analyte recognition based on its intrinsic behavior without using any labeling indicator. This not only improves the detection reliability, but also reduces sample preparation time and complexity involved during labeling step. Label-free biosensor devices are capable of monitoring analyte physiochemical properties such as binding sensitivity and selectivity, affinity constants and other dynamics of molecular recognition. The interface of a typical biosensor could range from natural antibodies to synthetic receptors for example molecular imprinted polymers (MIPs). The foremost advantages of using MIPs are their high binding selectivity comparable to natural antibodies, straightforward synthesis in short time, high thermal/chemical stability and compatibility with different transducers. Quartz crystal microbalance (QCM) resonators are leading acoustic devices that are extensively used for mass-sensitive measurements. Highlight features of QCM devices include low cost fabrication, room temperature operation, and most importantly ability to monitor extremely low mass shifts, thus potentially a universal transducer. The combination of MIPs with quartz QCM has turned out as a prominent sensing system for label-free recognition of diverse bioanalytes. In this article, we shall encompass the potential applications of MIP-QCM sensors exclusively label-free recognition of bacteria and virus species as representative micro and nanosized bioanalytes.

Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone.

PubMed

Singh, Vijay; Wang, Shenliang; Chan, Ke Min; Clark, Spencer A; Kool, Eric T

2013-04-24

Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.

Evaluating the compatibility of multi-functional and intensive urban land uses

NASA Astrophysics Data System (ADS)

Taleai, M.; Sharifi, A.; Sliuzas, R.; Mesgari, M.

2007-12-01

This research is aimed at developing a model for assessing land use compatibility in densely built-up urban areas. In this process, a new model was developed through the combination of a suite of existing methods and tools: geographical information system, Delphi methods and spatial decision support tools: namely multi-criteria evaluation analysis, analytical hierarchy process and ordered weighted average method. The developed model has the potential to calculate land use compatibility in both horizontal and vertical directions. Furthermore, the compatibility between the use of each floor in a building and its neighboring land uses can be evaluated. The method was tested in a built-up urban area located in Tehran, the capital city of Iran. The results show that the model is robust in clarifying different levels of physical compatibility between neighboring land uses. This paper describes the various steps and processes of developing the proposed land use compatibility evaluation model (CEM).

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

NASA Astrophysics Data System (ADS)

Wang, Taejun; Jang, Won Hyuk; Lee, Seunghun; Yoon, Calvin J.; Lee, Jun Ho; Kim, Bumju; Hwang, Sekyu; Hong, Chun-Pyo; Yoon, Yeoreum; Lee, Gilgu; Le, Viet-Hoan; Bok, Seoyeon; Ahn, G.-One; Lee, Jaewook; Gho, Yong Song; Chung, Euiheon; Kim, Sungjee; Jang, Myoung Ho; Myung, Seung-Jae; Kim, Myoung Joon; So, Peter T. C.; Kim, Ki Hean

2016-06-01

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

Visualization procedures for proteins and peptides on flat-bed monoliths and their effects on matrix-assisted laser-desorption/ionization time-of-flight mass spectrometric detection.

PubMed

Wouters, Bert; Vanhoutte, Dominique J D; Aarnoutse, Petra; Visser, Adriaan; Stassen, Catherine; Devreese, Bart; Kok, Wim Th; Schoenmakers, Peter J; Eeltink, Sebastiaan

2013-04-19

The present study concerns the application of visualization methods, i.e. coomassie-brilliant-blue-R staining (CBB-R), silver-nitrate staining, and fluorescamine labeling, and subsequent MALDI-MS analysis of intact proteins and peptides on the surface of flat-bed monoliths, intended for spatial two-dimensional chromatographic separations. The use of 100-μm thick macroporous poly(butyl methacrylate-co-ethylene dimethacrylate) flat-bed monoliths renders a fixation step obsolete, so that CBB-R and silver-nitrate staining and destaining could be achieved in 10-15 min as opposed to up to 24h, as is typical on 2D-PAGE gels. The detection limits remained comparable. The compatibility of the monolithic layer with subsequent MALDI-MS analysis of individual proteins and peptide spots was investigated with regards to mass accuracy, mass precision, resolution, and signal intensity. When comparing results from MALDI-MS analysis of proteins and peptides on a flat-bed monolith to results obtained directly on stainless-steel target plates, significant losses in mass precision, signal intensity, and an increased variation in resolution were observed. In addition, a loss in signal intensity up to two orders of magnitude was observed when using monolithic layers. After CCB-R and silver-nitrate staining and destaining to disrupt the protein-dye complexes no MALDI spectra with significant S/N ratios could be achieved. After fluorescamine labeling heterogeneous signals were observed, which resulted from a distribution in the number of fluorescence-labeled lysine groups and from the presence of labeled derivatives that had undergone condensation reactions. Copyright © 2013 Elsevier B.V. All rights reserved.

Utility of routinely acquired primary care data for paediatric disease epidemiology and pharmacoepidemiology

PubMed Central

Helms, Peter J; Daukes, Suzie Ekins; Taylor, Michael W; Simpson, Colin R; McLay, James S

2005-01-01

Background The majority of medicines prescribed for children are prescribed in primary care for common acute and chronic conditions. This is in contrast to prescribing in secondary care where the population of children admitted is small but where a large number of different medicines are prescribed to treat more serious and less common conditions. Methods Data on prescribing was extracted from the General Practice Administration System for Scotland (GPASS) for the year November 1999 to October 2000 and prescribing patterns for children aged 0–16 years expressed as percentages. A comparison of age specific consultations for asthma, as an example of a common paediatric condition, was also made between two separate general practice data sets, the General Practice Research Database (GRPD) and the continuous morbidity recording (CMR) subset of GPASS. Results Of 214 medicines investigated for unlicensed and off-label prescribing no unlicensed prescribing was identified. Off-label prescribing due to age was most common among younger and older children. The most common reasons for off-label prescriptions were, in order of frequency, lower than recommended dose, higher than recommended dose, below the recommended age, and unlicensed formulation. Age and gender specific consultations for asthma were similar in the two representative databases, GPRD and CMR, both showing disappearance of the male predominance in the teenage years. Conclusions Large primary care data sets available within a unified health care system such as the UK National Health Service (NHS) are likely to be broadly compatible and produce similar results. The prescribing of off-label medicines to children is common in primary care, most commonly due to prescribing out with the recommended dosage regimen. PMID:15948933

Culture of somatic cells isolated from frozen-thawed equine semen using fluorescence-assisted cell sorting.

PubMed

Brom-de-Luna, Joao Gatto; Canesin, Heloísa Siqueira; Wright, Gus; Hinrichs, Katrin

2018-03-01

Nuclear transfer using somatic cells from frozen semen (FzSC) would allow cloning of animals for which no other genetic material is available. Horses are one of the few species for which cloning is commercially feasible; despite this, there is no information available on the culture of equine FzSC. After preliminary trials on equine FzSC, recovered by density-gradient centrifugation, resulted in no growth, we hypothesized that sperm in the culture system negatively affected cell proliferation. Therefore, we evaluated culture of FzSC isolated using fluorescence-assisted cell sorting. In Exp. 1, sperm were labeled using antibodies to a sperm-specific antigen, SP17, and unlabeled cells were collected. This resulted in high sperm contamination. In Exp. 2, FzSC were labeled using an anti-MHC class I antibody. This resulted in an essentially pure population of FzSC, 13-25% of which were nucleated. Culture yielded no proliferation in any of nine replicates. In Exp. 3, 5 × 10 3 viable fresh, cultured horse fibroblasts were added to the frozen-thawed, washed semen, then this suspension was labeled and sorted as for Exp. 2. The enriched population had a mean of five sperm per recovered somatic cell; culture yielded formation of monolayers. In conclusion, an essentially pure population of equine FzSC could be obtained using sorting for presence of MHC class I antigens. No equine FzSC grew in culture; however, the proliferation of fibroblasts subjected to the same processing demonstrated that the labeling and sorting methods, and the presence of few sperm in culture, were compatible with cell viability. Copyright © 2017 Elsevier B.V. All rights reserved.

Osmoregulation in Methanogens (and Other Interesting Organisms)

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

Roberts, Mary Fedarko

2014-12-03

Our research has been aimed at (i) identifying, (ii) determining mode of regulation, and (iii) understanding how different classes of compatible solutes (also termed osmolytes) affect macromolecular stability in response to osmotic and thermal stress. For solutes we have identified (e.g., di-inositol-1,1’-phosphate (DIP)), we used NMR to elucidate biosynthetic pathways and then cloned suspected enzymes in the pathway to explore how they are regulated. Compatible solutes are thought to protect proteins from thermal and osmotic stresses by being excluded from the surface, allowing critical water molecules to interact with the protein. This implies there are no specific binding interactions betweenmore » osmolytes and proteins. However, we and others have often observed very specific solute effects for proteins that suggest a more direct interaction between solute and protein is likely can occur. Measuring such a weak interaction is extremely difficult. We have developed a solution NMR method, high-resolution field cycling relaxometry, that can measure spin-lattice relaxation rates as a function of magnetic field from 11.7 (the field of a 500 MHz spectrometer) to 0.003 T. The methodology is ideal for nuclei in small molecules with moderately long relaxation times at high fields – phosphate groups (31P), enriched carbonyls (13C), or methyl groups (1H). The protein of interest is spin-labeled to introduce a large dipole on it that will dominate the relaxation of nuclei on any small molecules that bind transiently. The key is to measure relaxation below 1-2 T (and extract nuclei-spin label distances in the bound complex) where the small molecule relaxation will be dominated by dipolar mechanisms with a correlation time indicative of the large protein complex. Our explorations of an inositol monophosphatase (the last step in DIP generation) localized four discrete binding sides for the thermoprotectant α-glutamate. This is a novel approach, and while the work did not fully explain how this solute protected the IMPase from thermal denaturation, it did showcase a new and exciting method to monitor weak binding in biological systems.« less

An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies

PubMed Central

Sun, Ning; Shibata, Brad; Hess, John F.

2015-01-01

Purpose Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques. Methods A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed. Results An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence. Conclusions A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic. PMID:25991907

Label-free SnO2 nanowire FET biosensor for protein detection

NASA Astrophysics Data System (ADS)

Jakob, Markus H.; Dong, Bo; Gutsch, Sebastian; Chatelle, Claire; Krishnaraja, Abinaya; Weber, Wilfried; Zacharias, Margit

2017-06-01

Novel tin oxide field-effect-transistors (SnO2 NW-FET) for pH and protein detection applicable in the healthcare sector are reported. With a SnO2 NW-FET the proof-of-concept of a bio-sensing device is demonstrated using the carrier transport control of the FET channel by a (bio-) liquid modulated gate. Ultra-thin Al2O3 fabricated by a low temperature atomic layer deposition (ALD) process represents a sensitive layer to H+ ions safeguarding the nanowire at the same time. Successful pH sensitivity is demonstrated for pH ranging from 3 to 10. For protein detection, the SnO2 NW-FET is functionalized with a receptor molecule which specifically interacts with the protein of interest to be detected. The feasibility of this approach is demonstrated via the detection of a biotinylated protein using a NW-FET functionalized with streptavidin. An immediate label-free electronic read-out of the signal is shown. The well-established Enzyme-Linked Immunosorbent Assay (ELISA) method is used to determine the optimal experimental procedure which would enable molecular binding events to occur while being compatible with a final label-free electronic read-out on a NW-FET. Integration of the bottom-up fabricated SnO2 NW-FET pH- and biosensor into a microfluidic system (lab-on-a-chip) allows the automated analysis of small volumes in the 400 μl range as would be desired in portable on-site point-of-care (POC) devices for medical diagnosis.

A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes

PubMed Central

Wang, Dan Ohtan; Matsuno, Hitomi; Ikeda, Shuji; Nakamura, Akiko; Yanagisawa, Hiroyuki; Hayashi, Yasunori; Okamoto, Akimitsu

2012-01-01

Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO–FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO–FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO–FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO–FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution. PMID:22101241

Use of Protein Biotinylation In Vivo for Immunoelectron Microscopic Localization of a Specific Protein Isoform

PubMed Central

Viens, Antoine; Harper, Francis; Pichard, Evelyne; Comisso, Martine; Pierron, Gérard; Ogryzko, Vasily

2008-01-01

Tagging of proteins in vivo by covalent attachment of a biotin moiety has emerged as a new prospective tool for protein detection and purification. Previously, we established a strategy for expression of in vivo biotinylated proteins in mammalian cells. It is based on coexpression of the protein of interest fused to a short biotin acceptor peptide and biotin ligase BirA cloned in the same vector. We show here that the in vivo biotinylation can be used for immunogold postembedding labeling in immunoelectron microscopy experiments. We show that immunoelectron microscopy with biotinylated nuclear proteins is compatible with a wide range of postembedding methods, facilitating combination of morphological and localization studies in a single experiment. We also show that the method works in both transient transfection and stable cell line expression protocols and can be used for colocalization studies. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:911–919, 2008) PMID:18574249

Measuring APC/C-Dependent Ubiquitylation In Vitro.

PubMed

Jarvis, Marc A; Brown, Nicholas G; Watson, Edmond R; VanderLinden, Ryan; Schulman, Brenda A; Peters, Jan-Michael

2016-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.

Designing deoxidation inhibiting encapsulation of metal oxide nanostructures for fluidic and biological applications

NASA Astrophysics Data System (ADS)

Ghosh, Moumita; Ghosh, Siddharth; Seibt, Michael; Schaap, Iwan A. T.; Schmidt, Christoph F.; Mohan Rao, G.

2016-12-01

Due to their photoluminescence, metal oxide nanostructures such as ZnO nanostructures are promising candidates in biomedical imaging, drug delivery and bio-sensing. To apply them as label for bio-imaging, it is important to study their structural stability in a bio-fluidic environment. We have explored the effect of water, the main constituent of biological solutions, on ZnO nanostructures with scanning electron microscopy (SEM) and photoluminescence (PL) studies which show ZnO nanorod degeneration in water. In addition, we propose and investigate a robust and inexpensive method to encapsulate these nanostructures (without structural degradation) using bio-compatible non-ionic surfactant in non-aqueous medium, which was not reported earlier. This new finding is an immediate interest to the broad audience of researchers working in biophysics, sensing and actuation, drug delivery, food and cosmetics technology, etc.

CHEMICAL STORAGE: MYTHS VERSUS REALITY

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

Simmons, F

A large number of resources explaining proper chemical storage are available. These resources include books, databases/tables, and articles that explain various aspects of chemical storage including compatible chemical storage, signage, and regulatory requirements. Another source is the chemical manufacturer or distributor who provides storage information in the form of icons or color coding schemes on container labels. Despite the availability of these resources, chemical accidents stemming from improper storage, according to recent reports (1) (2), make up almost 25% of all chemical accidents. This relatively high percentage of chemical storage accidents suggests that these publications and color coding schemes althoughmore » helpful, still provide incomplete information that may not completely mitigate storage risks. This manuscript will explore some ways published storage information may be incomplete, examine the associated risks, and suggest methods to help further eliminate chemical storage risks.« less

Compatible Spatial Discretizations for Partial Differential Equations

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

Arnold, Douglas, N, ed.

From May 11--15, 2004, the Institute for Mathematics and its Applications held a hot topics workshop on Compatible Spatial Discretizations for Partial Differential Equations. The numerical solution of partial differential equations (PDE) is a fundamental task in science and engineering. The goal of the workshop was to bring together a spectrum of scientists at the forefront of the research in the numerical solution of PDEs to discuss compatible spatial discretizations. We define compatible spatial discretizations as those that inherit or mimic fundamental properties of the PDE such as topology, conservation, symmetries, and positivity structures and maximum principles. A wide varietymore » of discretization methods applied across a wide range of scientific and engineering applications have been designed to or found to inherit or mimic intrinsic spatial structure and reproduce fundamental properties of the solution of the continuous PDE model at the finite dimensional level. A profusion of such methods and concepts relevant to understanding them have been developed and explored: mixed finite element methods, mimetic finite differences, support operator methods, control volume methods, discrete differential forms, Whitney forms, conservative differencing, discrete Hodge operators, discrete Helmholtz decomposition, finite integration techniques, staggered grid and dual grid methods, etc. This workshop seeks to foster communication among the diverse groups of researchers designing, applying, and studying such methods as well as researchers involved in practical solution of large scale problems that may benefit from advancements in such discretizations; to help elucidate the relations between the different methods and concepts; and to generally advance our understanding in the area of compatible spatial discretization methods for PDE. Particular points of emphasis included: + Identification of intrinsic properties of PDE models that are critical for the fidelity of numerical simulations. + Identification and design of compatible spatial discretizations of PDEs, their classification, analysis, and relations. + Relationships between different compatible spatial discretization methods and concepts which have been developed; + Impact of compatible spatial discretizations upon physical fidelity, verification and validation of simulations, especially in large-scale, multiphysics settings. + How solvers address the demands placed upon them by compatible spatial discretizations. This report provides information about the program and abstracts of all the presentations.« less

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

PubMed Central

2011-01-01

Background Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. Results An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). Conclusions The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy. PMID:21864341

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence.

PubMed

Sobolewski, Ireneusz; Polska, Katarzyna; Zylicz-Stachula, Agnieszka; Jeżewska-Frąckowiak, Joanna; Rak, Janusz; Skowron, Piotr

2011-08-24

Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.

ReagentTF: a rapid and versatile optical clearing method for biological imaging(Conference Presentation)

NASA Astrophysics Data System (ADS)

Yu, Tingting; Zhu, Jingtan; Li, Yusha; Qi, Yisong; Xu, Jianyi; Gong, Hui; Luo, Qingming; Zhu, Dan

2017-02-01

The emergence of various optical clearing methods provides a great potential for imaging deep inside tissues by combining with multiple-labelling and microscopic imaging techniques. They were generally developed for specific imaging demand thus presented some non-negligible limitations such as long incubation time, tissue deformation, fluorescence quenching, incompatibility with immunostaining or lipophilic tracers. In this study, we developed a rapid and versatile clearing method, termed ReagentTF, for deep imaging of various fluorescent samples. This method can not only efficiently clear embryos, neonatal whole-brains and adult thick brain sections by simple immersion in aqueous mixtures with minimal volume change, but also can preserve fluorescence of various fluorescent proteins and simultaneously be compatible with immunostaining and lipophilic neuronal dyes. We demonstrate the effectiveness of this method in reconstructing the cell distributions of mouse hippocampus, visualizing the neural projection from CA1 (Cornu Ammonis 1) to HDB (nucleus of the horizontal limb of the diagonal band), and observing the growth of forelimb plexus in whole-mount embryos. These results suggest that ReagentTF is useful for large-volume imaging and will be an option for the deep imaging of biological tissues.

Lectin-binding histochemistry of non-decalcified growth plate cartilage: a postembedment method for light microscopy of epon-embedded tissue.

PubMed

Farnum, C E; Wilsman, N J

1984-06-01

A postembedment method for the localization of lectin-binding glycoconjugates was developed using Epon-embedded growth plate cartilage from Yucatan miniature swine. By testing a variety of etching, blocking, and incubation procedures, a standard protocol was developed for 1 micron thick sections that allowed visualization of both intracellular and extracellular glycoconjugates with affinity for wheat germ agglutinin and concanavalin A. Both fluorescent and peroxidase techniques were used, and comparisons were made between direct methods and indirect methods using the biotin-avidin bridging system. Differential extracellular lectin binding allowed visualization of interterritorial , territorial, and pericellular matrices. Double labeling experiments showed the precision with which intracellular binding could be localized to specific cytoplasmic compartments, with resolution of binding to the Golgi apparatus, endoplasmic reticulum, and nuclear membrane at the light microscopic level. This method allows the localization of both intracellular and extracellular lectin-binding glycoconjugates using fixation and embedment procedures that are compatible with simultaneous ultrastructural analysis. As such it should have applicability both to the morphological analysis of growth plate organization during normal endochondral ossification, as well as to the diagnostic pathology of matrix abnormalities in disease states of growing cartilage.

High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

Preservation of three-dimensional spatial structure in the gut microbiome.

PubMed

Hasegawa, Yuko; Mark Welch, Jessica L; Rossetti, Blair J; Borisy, Gary G

2017-01-01

Preservation of three-dimensional structure in the gut is necessary in order to analyze the spatial organization of the gut microbiota and gut luminal contents. In this study, we evaluated preparation methods for mouse gut with the goal of preserving micron-scale spatial structure while performing fluorescence imaging assays. Our evaluation of embedding methods showed that commonly used media such as Tissue-Tek Optimal Cutting Temperature (OCT) compound, paraffin, and polyester waxes resulted in redistribution of luminal contents. By contrast, a hydrophilic methacrylate resin, Technovit H8100, preserved three-dimensional organization. Our mouse intestinal preparation protocol optimized using the Technovit H8100 embedding method was compatible with microbial fluorescence in situ hybridization (FISH) and other labeling techniques, including immunostaining and staining with both wheat germ agglutinin (WGA) and 4', 6-diamidino-2-phenylindole (DAPI). Mucus could be visualized whether the sample was fixed with paraformaldehyde (PFA) or with Carnoy's fixative. The protocol optimized in this study enabled simultaneous visualization of micron-scale spatial patterns formed by microbial cells in the mouse intestines along with biogeographical landmarks such as host-derived mucus and food particles.

Ultra-small dye-doped silica nanoparticles via modified sol-gel technique

NASA Astrophysics Data System (ADS)

Riccò, R.; Nizzero, S.; Penna, E.; Meneghello, A.; Cretaio, E.; Enrichi, F.

2018-05-01

In modern biosensing and imaging, fluorescence-based methods constitute the most diffused approach to achieve optimal detection of analytes, both in solution and on the single-particle level. Despite the huge progresses made in recent decades in the development of plasmonic biosensors and label-free sensing techniques, fluorescent molecules remain the most commonly used contrast agents to date for commercial imaging and detection methods. However, they exhibit low stability, can be difficult to functionalise, and often result in a low signal-to-noise ratio. Thus, embedding fluorescent probes into robust and bio-compatible materials, such as silica nanoparticles, can substantially enhance the detection limit and dramatically increase the sensitivity. In this work, ultra-small fluorescent silica nanoparticles (NPs) for optical biosensing applications were doped with a fluorescent dye, using simple water-based sol-gel approaches based on the classical Stöber procedure. By systematically modulating reaction parameters, controllable size tuning of particle diameters as low as 10 nm was achieved. Particles morphology and optical response were evaluated showing a possible single-molecule behaviour, without employing microemulsion methods to achieve similar results. [Figure not available: see fulltext.

Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections.

PubMed

Wegner, Kyle A; Keikhosravi, Adib; Eliceiri, Kevin W; Vezina, Chad M

2017-08-01

The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.

Inter-labeler and intra-labeler variability of condition severity classification models using active and passive learning methods.

PubMed

Nissim, Nir; Shahar, Yuval; Elovici, Yuval; Hripcsak, George; Moskovitch, Robert

2017-09-01

Labeling instances by domain experts for classification is often time consuming and expensive. To reduce such labeling efforts, we had proposed the application of active learning (AL) methods, introduced our CAESAR-ALE framework for classifying the severity of clinical conditions, and shown its significant reduction of labeling efforts. The use of any of three AL methods (one well known [SVM-Margin], and two that we introduced [Exploitation and Combination_XA]) significantly reduced (by 48% to 64%) condition labeling efforts, compared to standard passive (random instance-selection) SVM learning. Furthermore, our new AL methods achieved maximal accuracy using 12% fewer labeled cases than the SVM-Margin AL method. However, because labelers have varying levels of expertise, a major issue associated with learning methods, and AL methods in particular, is how to best to use the labeling provided by a committee of labelers. First, we wanted to know, based on the labelers' learning curves, whether using AL methods (versus standard passive learning methods) has an effect on the Intra-labeler variability (within the learning curve of each labeler) and inter-labeler variability (among the learning curves of different labelers). Then, we wanted to examine the effect of learning (either passively or actively) from the labels created by the majority consensus of a group of labelers. We used our CAESAR-ALE framework for classifying the severity of clinical conditions, the three AL methods and the passive learning method, as mentioned above, to induce the classifications models. We used a dataset of 516 clinical conditions and their severity labeling, represented by features aggregated from the medical records of 1.9 million patients treated at Columbia University Medical Center. We analyzed the variance of the classification performance within (intra-labeler), and especially among (inter-labeler) the classification models that were induced by using the labels provided by seven labelers. We also compared the performance of the passive and active learning models when using the consensus label. The AL methods: produced, for the models induced from each labeler, smoother Intra-labeler learning curves during the training phase, compared to the models produced when using the passive learning method. The mean standard deviation of the learning curves of the three AL methods over all labelers (mean: 0.0379; range: [0.0182 to 0.0496]), was significantly lower (p=0.049) than the Intra-labeler standard deviation when using the passive learning method (mean: 0.0484; range: [0.0275-0.0724). Using the AL methods resulted in a lower mean Inter-labeler AUC standard deviation among the AUC values of the labelers' different models during the training phase, compared to the variance of the induced models' AUC values when using passive learning. The Inter-labeler AUC standard deviation, using the passive learning method (0.039), was almost twice as high as the Inter-labeler standard deviation using our two new AL methods (0.02 and 0.019, respectively). The SVM-Margin AL method resulted in an Inter-labeler standard deviation (0.029) that was higher by almost 50% than that of our two AL methods The difference in the inter-labeler standard deviation between the passive learning method and the SVM-Margin learning method was significant (p=0.042). The difference between the SVM-Margin and Exploitation method was insignificant (p=0.29), as was the difference between the Combination_XA and Exploitation methods (p=0.67). Finally, using the consensus label led to a learning curve that had a higher mean intra-labeler variance, but resulted eventually in an AUC that was at least as high as the AUC achieved using the gold standard label and that was always higher than the expected mean AUC of a randomly selected labeler, regardless of the choice of learning method (including a passive learning method). Using a paired t-test, the difference between the intra-labeler AUC standard deviation when using the consensus label, versus that value when using the other two labeling strategies, was significant only when using the passive learning method (p=0.014), but not when using any of the three AL methods. The use of AL methods, (a) reduces intra-labeler variability in the performance of the induced models during the training phase, and thus reduces the risk of halting the process at a local minimum that is significantly different in performance from the rest of the learned models; and (b) reduces Inter-labeler performance variance, and thus reduces the dependence on the use of a particular labeler. In addition, the use of a consensus label, agreed upon by a rather uneven group of labelers, might be at least as good as using the gold standard labeler, who might not be available, and certainly better than randomly selecting one of the group's individual labelers. Finally, using the AL methods: when provided by the consensus label reduced the intra-labeler AUC variance during the learning phase, compared to using passive learning. Copyright © 2017 Elsevier B.V. All rights reserved.

Inter-Labeler and Intra-Labeler Variability of Condition Severity Classification Models Using Active and Passive Learning Methods

PubMed Central

Nissim, Nir; Shahar, Yuval; Boland, Mary Regina; Tatonetti, Nicholas P; Elovici, Yuval; Hripcsak, George; Moskovitch, Robert

2018-01-01

Background and Objectives Labeling instances by domain experts for classification is often time consuming and expensive. To reduce such labeling efforts, we had proposed the application of active learning (AL) methods, introduced our CAESAR-ALE framework for classifying the severity of clinical conditions, and shown its significant reduction of labeling efforts. The use of any of three AL methods (one well known [SVM-Margin], and two that we introduced [Exploitation and Combination_XA]) significantly reduced (by 48% to 64%) condition labeling efforts, compared to standard passive (random instance-selection) SVM learning. Furthermore, our new AL methods achieved maximal accuracy using 12% fewer labeled cases than the SVM-Margin AL method. However, because labelers have varying levels of expertise, a major issue associated with learning methods, and AL methods in particular, is how to best to use the labeling provided by a committee of labelers. First, we wanted to know, based on the labelers’ learning curves, whether using AL methods (versus standard passive learning methods) has an effect on the Intra-labeler variability (within the learning curve of each labeler) and inter-labeler variability (among the learning curves of different labelers). Then, we wanted to examine the effect of learning (either passively or actively) from the labels created by the majority consensus of a group of labelers. Methods We used our CAESAR-ALE framework for classifying the severity of clinical conditions, the three AL methods and the passive learning method, as mentioned above, to induce the classifications models. We used a dataset of 516 clinical conditions and their severity labeling, represented by features aggregated from the medical records of 1.9 million patients treated at Columbia University Medical Center. We analyzed the variance of the classification performance within (intra-labeler), and especially among (inter-labeler) the classification models that were induced by using the labels provided by seven labelers. We also compared the performance of the passive and active learning models when using the consensus label. Results The AL methods produced, for the models induced from each labeler, smoother Intra-labeler learning curves during the training phase, compared to the models produced when using the passive learning method. The mean standard deviation of the learning curves of the three AL methods over all labelers (mean: 0.0379; range: [0.0182 to 0.0496]), was significantly lower (p = 0.049) than the Intra-labeler standard deviation when using the passive learning method (mean: 0.0484; range: [0.0275 to 0.0724). Using the AL methods resulted in a lower mean Inter-labeler AUC standard deviation among the AUC values of the labelers’ different models during the training phase, compared to the variance of the induced models’ AUC values when using passive learning. The Inter-labeler AUC standard deviation, using the passive learning method (0.039), was almost twice as high as the Inter-labeler standard deviation using our two new AL methods (0.02 and 0.019, respectively). The SVM-Margin AL method resulted in an Inter-labeler standard deviation (0.029) that was higher by almost 50% than that of our two AL methods. The difference in the inter-labeler standard deviation between the passive learning method and the SVM-Margin learning method was significant (p = 0.042). The difference between the SVM-Margin and Exploitation method was insignificant (p = 0.29), as was the difference between the Combination_XA and Exploitation methods (p = 0.67). Finally, using the consensus label led to a learning curve that had a higher mean intra-labeler variance, but resulted eventually in an AUC that was at least as high as the AUC achieved using the gold standard label and that was always higher than the expected mean AUC of a randomly selected labeler, regardless of the choice of learning method (including a passive learning method). Using a paired t-test, the difference between the intra-labeler AUC standard deviation when using the consensus label, versus that value when using the other two labeling strategies, was significant only when using the passive learning method (p = 0.014), but not when using any of the three AL methods. Conclusions The use of AL methods, (a) reduces intra-labeler variability in the performance of the induced models during the training phase, and thus reduces the risk of halting the process at a local minimum that is significantly different in performance from the rest of the learned models; and (b) reduces Inter-labeler performance variance, and thus reduces the dependence on the use of a particular labeler. In addition, the use of a consensus label, agreed upon by a rather uneven group of labelers, might be at least as good as using the gold standard labeler, who might not be available, and certainly better than randomly selecting one of the group’s individual labelers. Finally, using the AL methods when provided by the consensus label reduced the intra-labeler AUC variance during the learning phase, compared to using passive learning. PMID:28456512

Analysis of lipid raft molecules in the living brain slices.

PubMed

Kotani, Norihiro; Nakano, Takanari; Ida, Yui; Ito, Rina; Hashizume, Miki; Yamaguchi, Arisa; Seo, Makoto; Araki, Tomoyuki; Hojo, Yasushi; Honke, Koichi; Murakoshi, Takayuki

2017-08-24

Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the "Enzyme-Mediated Activation of Radical Sources (EMARS)" method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called "EMARS products" distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Real-time label-free biosensing with integrated planar waveguide ring resonators

NASA Astrophysics Data System (ADS)

Sohlström, Hans; Gylfason, Kristinn B.; Hill, Daniel

2010-05-01

We review the use of planar integrated optical waveguide ring resonators for label free bio-sensing and present recent results from two European biosensor collaborations: SABIO and InTopSens. Planar waveguide ring resonators are attractive for label-free biosensing due to their small footprint, high Q-factors, and compatibility with on-chip optics and microfluidics. This enables integrated sensor arrays for compact labs-on-chip. One application of label-free sensor arrays is for point-of-care medical diagnostics. Bringing such powerful tools to the single medical practitioner is an important step towards personalized medicine, but requires addressing a number of issues: improving limit of detection, managing the influence of temperature, parallelization of the measurement for higher throughput and on-chip referencing, efficient light-coupling strategies to simplify alignment, and packaging of the optical chip and integration with microfluidics. From the SABIO project we report refractive index measurement and label-free biosensing in an 8-channel slotwaveguide ring resonator sensor array, within a compact cartridge with integrated microfluidics. The sensors show a volume sensing detection limit of 5 x 10-6 RIU and a surface sensing detection limit of 0.9 pg/mm2. From the InTopSens project we report early results on silicon-on-insulator racetrack resonators.

Self-(in)compatibility of the almonds P. dulcis and P. webbii: detection and cloning of 'wild-type Sf ' and new self-compatibility alleles encoding inactive S-RNases.

PubMed

Bosković, Radovan I; Tobutt, Kenneth R; Ortega, Encarnación; Sutherland, Bruce G; Godini, Angelo

2007-12-01

Prunus dulcis, the almond, is a predominantly self-incompatible (SI) species with a gametophytic self-incompatibility system mediated by S-RNases. The economically important allele Sf, which results in self-compatibility in P. dulcis, is said to have arisen by introgression from Prunus webbii in the Italian region of Apulia. We investigated the range of self-(in)compatibility alleles in Apulian material of the two species. About 23 cultivars of P. dulcis (14 self-compatible (SC) and nine SI) and 33 accessions of P. webbii (16 SC, two SI and 15 initially of unknown status), all from Apulia, were analysed using PCR of genomic DNA to amplify S-RNase alleles and, in most cases, IEF and staining of stylar protein extracts to detect S-RNase activity. Some amplification products were cloned and sequenced. The allele Sf was present in nearly all the SC cultivars of P. dulcis but, surprisingly, was absent from nearly all SC accessions of P. webbii. And of particular interest was the presence in many SI cultivars of P. dulcis of a new active allele, labelled S30, the sequence of which showed it to be the wild-type of Sf so that Sf can be regarded as a stylar part mutant S30 degrees . These findings indicate Sf may have arisen within P. dulcis, by mutation. One SC cultivar of P. dulcis, 'Patalina', had a new self-compatibility allele lacking RNase activity, Sn5, which could be useful in breeding programmes. In the accessions of P. webbii, some of which were known to be SC, three new alleles were found which lacked RNase activity but had normal DNA sequences.

MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Presley, Andrew D; Fuller, Kathryn M; Arriaga, Edgar A

2003-08-05

MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.

Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

PubMed

Paradh, A D; Hill, A E; Mitchell, W J

2014-01-01

DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories. © 2013 Elsevier B.V. All rights reserved.

Integrated Force Method for Indeterminate Structures

NASA Technical Reports Server (NTRS)

Hopkins, Dale A.; Halford, Gary R.; Patnaik, Surya N.

2008-01-01

Two methods of solving indeterminate structural-mechanics problems have been developed as products of research on the theory of strain compatibility. In these methods, stresses are considered to be the primary unknowns (in contrast to strains and displacements being considered as the primary unknowns in some prior methods). One of these methods, denoted the integrated force method (IFM), makes it possible to compute stresses, strains, and displacements with high fidelity by use of modest finite-element models that entail relatively small amounts of computation. The other method, denoted the completed Beltrami Mitchell formulation (CBMF), enables direct determination of stresses in an elastic continuum with general boundary conditions, without the need to first calculate displacements as in traditional methods. The equilibrium equation, the compatibility condition, and the material law are the three fundamental concepts of the theory of structures. For almost 150 years, it has been commonly supposed that the theory is complete. However, until now, the understanding of the compatibility condition remained incomplete, and the compatibility condition was confused with the continuity condition. Furthermore, the compatibility condition as applied to structures in its previous incomplete form was inconsistent with the strain formulation in elasticity.

The functional architecture of the human body: assessing body representation by sorting body parts and activities.

PubMed

Bläsing, Bettina; Schack, Thomas; Brugger, Peter

2010-05-01

We investigated mental representations of body parts and body-related activities in two subjects with congenitally absent limbs (one with, the other without phantom sensations), a wheelchair sports group of paraplegic participants, and two groups of participants with intact limbs. To analyse mental representation structures, we applied Structure Dimensional Analysis. Verbal labels indicating body parts and related activities were presented in randomized lists that had to be sorted according to a hierarchical splitting paradigm. Participants were required to group the items according to whether or not they were considered related, based on their own body perception. Results of the groups of physically intact and paraplegic participants revealed separate clusters for the lower body, upper body, fingers and head. The participant with congenital phantom limbs also showed a clear separation between upper and lower body (but not between fingers and hands). In the participant without phantom sensations of the absent arms, no such modularity emerged, but the specific practice of his right foot in communication and daily routines was reflected. Sorting verbal labels of body parts and activities appears a useful method to assess body representation in individuals with special body anatomy or function and leads to conclusions largely compatible with other assessment procedures.

[INVITED] Surface plasmon cavities on optical fiber end-facets for biomolecule and ultrasound detection

NASA Astrophysics Data System (ADS)

Yang, Tian; He, Xiaolong; Zhou, Xin; Lei, Zeyu; Wang, Yalin; Yang, Jie; Cai, De; Chen, Sung-Liang; Wang, Xueding

2018-05-01

Integrating surface plasmon resonance (SPR) devices upon single-mode fiber (SMF) end facets renders label-free sensing systems that have a simple dip-and-read configuration, a small form factor, high compatibility with fiber-optic techniques, and invasive testing capability. Such devices are not only low cost replacement of current equipments in centralized laboratories, but also highly desirable for opening paths to new applications of label-free optical sensing technologies, such as point-of-care immunological tests and intravascular ultrasound imaging. In this paper, we explain the requirements and challenges for such devices from the perspectives of biomolecule and ultrasound detection applications. In such a context, we review our recent work on SMF end-facet SPR cavities. This include a glue-and-strip fabrication method to transfer a nano-patterned thin gold film to the SMF end-facet with high yield, high quality and high alignment precision, the designs of distributed Bragg reflector (DBR) and distributed feedback (DFB) SPR cavities that couple efficiently with the SMF guided mode and reach quality factors of over 100, and the preliminary results for biomolecule interaction sensing and ultrasound detection. The particular advantages and potential values of these devices have been discussed, in terms of sensitivity, data reliability, reproducibility, bandwidth, etc.

Synthesis and Characterization of a Magnetically Active 19F Molecular Beacon.

PubMed

Dempsey, Megan E; Marble, Hetal D; Shen, Tun-Li; Fawzi, Nicolas L; Darling, Eric M

2018-02-21

Gene expression is used extensively to describe cellular characteristics and behaviors; however, most methods of assessing gene expression are unsuitable for living samples, requiring destructive processes such as fixation or lysis. Recently, molecular beacons have become a viable tool for live-cell imaging of mRNA molecules in situ. Historically, beacon-mediated imaging has been limited to fluorescence-based approaches. We propose the design and synthesis of a novel molecular beacon for magnetic resonance detection of any desired target nucleotide sequence. The biologically compatible synthesis incorporates commonly used bioconjugation reactions in aqueous conditions and is accessible for laboratories without extensive synthesis capabilities. The resulting beacon uses fluorine ( 19 F) as a reporter, which is broadened, or turned "off", via paramagnetic relaxation enhancement from a stabilized nitroxide radical spin label when the beacon is not bound to its nucleic acid target. Therefore, the 19 F NMR signal of the beacon is quenched in its hairpin conformation when the spin label and the 19 F substituent are held in proximity, but the signal is recovered upon beacon hybridization to its specific complementary nucleotide sequence by physical separation of the radical from the 19 F reporter. This study establishes a path for magnetic resonance-based assessment of specific mRNA expression, providing new possibilities for applying molecular beacon technology in living systems.

Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

PubMed Central

Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C.; Min, Wei

2013-01-01

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo. PMID:23798434

Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

PubMed Central

2015-01-01

The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

PubMed Central

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-01-01

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

Detection and size analysis of proteins with switchable DNA layers.

PubMed

Rant, Ulrich; Pringsheim, Erika; Kaiser, Wolfgang; Arinaga, Kenji; Knezevic, Jelena; Tornow, Marc; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard

2009-04-01

We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody fragments, which can be detected in quantities of less than 10(-18) mol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.

Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries.

PubMed

Zimmermann, Gunther; Li, Yizhou; Rieder, Ulrike; Mattarella, Martin; Neri, Dario; Scheuermann, Jörg

2017-05-04

DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article we present new methods based on hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides; these facilitate the determination of affinity constants and kinetic dissociation constants. The experimental procedures were demonstrated with acetazolamide, a binder to carbonic anhydrase IX with a dissociation constant in the nanomolar range. The detection of binding events was compatible not only with fluorescence polarization methodologies, but also with Alphascreen technology and with microscale thermophoresis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative and microscopic assessment of compatible and incompatible interactions between chickpea cultivars and Fusarium oxysporum f. sp. ciceris races.

PubMed

Jiménez-Fernández, Daniel; Landa, Blanca B; Kang, Seogchan; Jiménez-Díaz, Rafael M; Navas-Cortés, Juan A

2013-01-01

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris, a main threat to global chickpea production, is managed mainly by resistant cultivars whose efficiency is curtailed by Fusarium oxysporum f. sp. ciceris races. We characterized compatible and incompatible interactions by assessing the spatial-temporal pattern of infection and colonization of chickpea cvs. P-2245, JG-62 and WR-315 by Fusarium oxysporum f. sp. ciceris races 0 and 5 labeled with ZsGreen fluorescent protein using confocal laser scanning microscopy. The two races colonized the host root surface in both interactions with preferential colonization of the root apex and subapical root zone. In compatible interactions, the pathogen grew intercellularly in the root cortex, reached the xylem, and progressed upwards in the stem xylem, being the rate and intensity of stem colonization directly related with the degree of compatibility among Fusarium oxysporum f. sp. ciceris races and chickpea cultivars. In incompatible interactions, race 0 invaded and colonized 'JG-62' xylem vessels of root and stem but in 'WR-315', it remained in the intercellular spaces of the root cortex failing to reach the xylem, whereas race 5 progressed up to the hypocotyl. However, all incompatible interactions were asymptomatic. The differential patterns of colonization of chickpea cultivars by Fusarium oxysporum f. sp. ciceris races may be related to the operation of multiple resistance mechanisms.

Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane anchorage region

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

Chakravarty, R.; Rice, R.H.

1989-01-05

The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of (/sup 3/H) acetic, (/sup 3/H)myristic, or (/sup 3/H)palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction ofmore » cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH/sub 2/ or COOH terminus.« less

Application of higher energy collisional dissociation (HCD) to the fragmentation of new DOTA-based labels and N-termini DOTA-labeled peptides.

PubMed

El-Khatib, A H; He, Y; Esteban-Fernández, D; Linscheid, M W

2017-08-01

1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives are applied in quantitative proteomics owing to their ability to react with different functional groups, to harbor lanthanoides and hence their compatibility with molecular and elemental mass spectrometry. The new DOTA derivatives, namely Ln-MeCAT-Click and Ln-DOTA-Dimedone, allow efficient thiol labeling and targeting sulfenation as an important post-translational modification, respectively. Quantitative applications require the investigation of fragmentation behavior of these reagents. Therefore, the fragmentation behavior of Ln-MeCAT-Click and Ln-DOTA-Dimedone was studied using collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and higher-energy collision dissociation (HCD) using different energy levels, and the efficiency of reporter ion production was estimated. The efficiency of characteristic fragment formation was in the order IRMPD > HCD (normal energy level) > CID. On the other hand, the application of HCD at high energy levels (HCD@HE; NCE > 250%) resulted in a significant increase in reporter ion production (33-54%). This new strategy was successfully applied to generate label-specific reporter ions for DOTA amino labeling at the N-termini and in a quantitative fashion for the estimation of amino:thiol ratio in peptides. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

High-throughput, low-loss, low-cost, and label-free cell separation using electrophysiology-activated cell enrichment.

PubMed

Faraghat, Shabnam A; Hoettges, Kai F; Steinbach, Max K; van der Veen, Daan R; Brackenbury, William J; Henslee, Erin A; Labeed, Fatima H; Hughes, Michael P

2017-05-02

Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell-separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whereas the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation), and cost. The system was evaluated using three example separations: live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells per second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 min, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells per second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells while maintaining a processing rate of ∼170,000 cells per second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where good manufacturing practice compatibility is of paramount importance.

SPRi-based biosensing platforms for detection of specific DNA sequences using thiolate and dithiocarbamate assemblies

NASA Astrophysics Data System (ADS)

Drozd, Marcin; Pietrzak, Mariusz D.; Malinowska, Elżbieta

2018-05-01

The framework of presented study covers the development and examination of the analytical performance of surface plasmon resonance-based (SPR) DNA biosensors dedicated for a detection of model target oligonucleotide sequence. For this aim, various strategies of immobilization of DNA probes on gold transducers were tested. Besides the typical approaches: chemisorption of thiolated ssDNA (DNA-thiol) and physisorption of non-functionalized oligonucleotides, relatively new method based on chemisorption of dithiocarbamate-functionalized ssDNA (DNA-DTC) was applied for the first time for preparation of DNA-based SPR biosensor. The special emphasis was put on the correlation between the method of DNA immobilization and the composition of obtained receptor layer. The carried out studies focused on the examination of the capability of developed receptors layers to interact with both target DNA and DNA-functionalized AuNPs. It was found, that the detection limit of target DNA sequence (27 nb length) depends on the strategy of probe immobilization and backfilling method, and in the best case it amounted to 0,66 nM. Moreover, the application of ssDNA-functionalized gold nanoparticles (AuNPs) as plasmonic labels for secondary enhancement of SPR response is presented. The influence of spatial organization and surface density of a receptor layer on the ability to interact with DNA-functionalized AuNPs is discussed. Due to the best compatibility of receptors immobilized via DTC chemisorption: 1.47 ± 0.4 ·1012 molecules • cm-2 (with the calculated area occupied by single nanoparticle label of 132.7 nm2), DNA chemisorption based on DTCs is pointed as especially promising for DNA biosensors utilizing indirect detection in competitive assays.

SPRi-Based Biosensing Platforms for Detection of Specific DNA Sequences Using Thiolate and Dithiocarbamate Assemblies.

PubMed

Drozd, Marcin; Pietrzak, Mariusz D; Malinowska, Elżbieta

2018-01-01

The framework of presented study covers the development and examination of the analytical performance of surface plasmon resonance-based (SPR) DNA biosensors dedicated for a detection of model target oligonucleotide sequence. For this aim, various strategies of immobilization of DNA probes on gold transducers were tested. Besides the typical approaches: chemisorption of thiolated ssDNA (DNA-thiol) and physisorption of non-functionalized oligonucleotides, relatively new method based on chemisorption of dithiocarbamate-functionalized ssDNA (DNA-DTC) was applied for the first time for preparation of DNA-based SPR biosensor. The special emphasis was put on the correlation between the method of DNA immobilization and the composition of obtained receptor layer. The carried out studies focused on the examination of the capability of developed receptors layers to interact with both target DNA and DNA-functionalized AuNPs. It was found, that the detection limit of target DNA sequence (27 nb length) depends on the strategy of probe immobilization and backfilling method, and in the best case it amounted to 0.66 nM. Moreover, the application of ssDNA-functionalized gold nanoparticles (AuNPs) as plasmonic labels for secondary enhancement of SPR response is presented. The influence of spatial organization and surface density of a receptor layer on the ability to interact with DNA-functionalized AuNPs is discussed. Due to the best compatibility of receptors immobilized via DTC chemisorption: 1.47 ± 0.4 · 10 12 molecules · cm -2 (with the calculated area occupied by single nanoparticle label of ~132.7 nm 2 ), DNA chemisorption based on DTCs is pointed as especially promising for DNA biosensors utilizing indirect detection in competitive assays.

Use of the method of biosphere compatibility for the assessment of environmental protection methods

NASA Astrophysics Data System (ADS)

Vorobyov, Sergey

2018-01-01

The article is devoted to the question of using the indicator of biosphere compatibility for assessing the effectiveness of environmental protection methods. The indicator of biosphere compatibility was proposed by the vice-president of RAASN (Russian Academy of Architecture and Building Sciences), Doctor of Technical Sciences, Professor V.I. Ilyichev. This indicator is allows not only qualitatively but also quantitatively to assess the degree of development of urban urban areas, from the standpoint of preserving the biosphere in urban ecosystems while realizing the city’s main functions. The integral indicator of biosphere compatibility is allows us to assess not only the current ecological situation in the territory under consideration, but also to plan the forecast of its changes for building the new construction projects, or for reconstructing existing ones. The indicator of biosphere compatibility, which is a mathematical expression of the tripartite balance (technosphere, biosphere and population of this area), is allows us to quantify the degree of effectiveness of different method of protecting the environment for choose the most effective for these conditions.

Integrated Force Method Solution to Indeterminate Structural Mechanics Problems

NASA Technical Reports Server (NTRS)

Patnaik, Surya N.; Hopkins, Dale A.; Halford, Gary R.

2004-01-01

Strength of materials problems have been classified into determinate and indeterminate problems. Determinate analysis primarily based on the equilibrium concept is well understood. Solutions of indeterminate problems required additional compatibility conditions, and its comprehension was not exclusive. A solution to indeterminate problem is generated by manipulating the equilibrium concept, either by rewriting in the displacement variables or through the cutting and closing gap technique of the redundant force method. Compatibility improvisation has made analysis cumbersome. The authors have researched and understood the compatibility theory. Solutions can be generated with equal emphasis on the equilibrium and compatibility concepts. This technique is called the Integrated Force Method (IFM). Forces are the primary unknowns of IFM. Displacements are back-calculated from forces. IFM equations are manipulated to obtain the Dual Integrated Force Method (IFMD). Displacement is the primary variable of IFMD and force is back-calculated. The subject is introduced through response variables: force, deformation, displacement; and underlying concepts: equilibrium equation, force deformation relation, deformation displacement relation, and compatibility condition. Mechanical load, temperature variation, and support settling are equally emphasized. The basic theory is discussed. A set of examples illustrate the new concepts. IFM and IFMD based finite element methods are introduced for simple problems.

Femtogram-scale photothermal spectroscopy of explosive molecules on nanostrings.

PubMed

Biswas, T S; Miriyala, N; Doolin, C; Liu, X; Thundat, T; Davis, J P

2014-11-18

We demonstrate detection of femtogram-scale quantities of the explosive molecule 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) via combined nanomechanical photothermal spectroscopy and mass desorption. Photothermal spectroscopy provides a spectroscopic fingerprint of the molecule, which is unavailable using mass adsorption/desorption alone. Our measurement, based on thermomechanical measurement of silicon nitride nanostrings, represents the highest mass resolution ever demonstrated via nanomechanical photothermal spectroscopy. This detection scheme is quick, label-free, and is compatible with parallelized molecular analysis of multicomponent targets.

Towards comprehensive structural motif mining for better fold annotation in the "twilight zone" of sequence dissimilarity

PubMed Central

Jia, Yi; Huan, Jun; Buhr, Vincent; Zhang, Jintao; Carayannopoulos, Leonidas N

2009-01-01

Background Automatic identification of structure fingerprints from a group of diverse protein structures is challenging, especially for proteins whose divergent amino acid sequences may fall into the "twilight-" or "midnight-" zones where pair-wise sequence identities to known sequences fall below 25% and sequence-based functional annotations often fail. Results Here we report a novel graph database mining method and demonstrate its application to protein structure pattern identification and structure classification. The biologic motivation of our study is to recognize common structure patterns in "immunoevasins", proteins mediating virus evasion of host immune defense. Our experimental study, using both viral and non-viral proteins, demonstrates the efficiency and efficacy of the proposed method. Conclusion We present a theoretic framework, offer a practical software implementation for incorporating prior domain knowledge, such as substitution matrices as studied here, and devise an efficient algorithm to identify approximate matched frequent subgraphs. By doing so, we significantly expanded the analytical power of sophisticated data mining algorithms in dealing with large volume of complicated and noisy protein structure data. And without loss of generality, choice of appropriate compatibility matrices allows our method to be easily employed in domains where subgraph labels have some uncertainty. PMID:19208148

Protein expression and isotopic enrichment based on induction of the Entner-Doudoroff pathway in Escherichia coli

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

Refaeli, Bosmat; Goldbourt, Amir, E-mail: amirgo@post.tau.ac.il

2012-10-12

Highlights: Black-Right-Pointing-Pointer The Entner-Doudoroff pathway is induced during protein expression in E. coli. Black-Right-Pointing-Pointer 1-{sup 13}C-gluconate and {sup 15}NH{sub 4}Cl provide a carbonyl-amide protein backbone labeling scheme. Black-Right-Pointing-Pointer The enrichment pattern is determined by nuclear magnetic resonance. -- Abstract: The Entner-Doudoroff pathway is known to exist in many organisms including bacteria, archea and eukarya. Although the common route for carbon catabolism in Escherichia coli is the Embden-Meyerhof-Parnas pathway, it was shown that gluconate catabolism in E. coli occurs via the Entner-Doudoroff pathway. We demonstrate here that by supplying BL21(DE3) competent E.coli cells with gluconate in a minimal growth medium, proteinmore » expression can be induced. Nuclear magnetic resonance data of over-expressed ubiquitin show that by using [1-{sup 13}C]-gluconate as the only carbon source, and {sup 15}N-enriched ammonium chloride, sparse isotopic enrichment in the form of a spin-pair carbonyl-amide backbone enrichment is obtained. The specific amino acid labeling pattern is analyzed and is shown to be compatible with Entner-Doudoroff metabolism. Isotopic enrichment serves as a key factor in the biophysical characterization of proteins by various methods including nuclear magnetic resonance, mass spectrometry, infrared spectroscopy and more. Therefore, the method presented here can be applied to study proteins by obtaining sparse enrichment schemes that are not based on the regular glycolytic pathway, or to study the Entner-Doudoroff metabolism during protein expression.« less

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

Compatibility Condition in Theory of Solid Mechanics (Elasticity, Structures, and Design Optimization)

NASA Technical Reports Server (NTRS)

Patnaik, Surya N.; Pai, Shantaram S.; Hopkins, Dale A.

2007-01-01

The strain formulation in elasticity and the compatibility condition in structural mechanics have neither been understood nor have they been utilized. This shortcoming prevented the formulation of a direct method to calculate stress. We have researched and understood the compatibility condition for linear problems in elasticity and in finite element analysis. This has lead to the completion of the method of force with stress (or stress resultant) as the primary unknown. The method in elasticity is referred to as the completed Beltrami-Michell formulation (CBMF), and it is the integrated force method (IFM) in structures. The dual integrated force method (IFMD) with displacement as the primary unknown has been formulated. IFM and IFMD produce identical responses. The variational derivation of the CBMF yielded the new boundary compatibility conditions. The CBMF can be used to solve stress, displacement, and mixed boundary value problems. The IFM in structures produced high-fidelity response even with a modest finite element model. The IFM has influenced structural design considerably. A fully utilized design method for strength and stiffness limitation has been developed. The singularity condition in optimization has been identified. The CBMF and IFM tensorial approaches are robust formulations because of simultaneous emphasis on the equilibrium equation and the compatibility condition.

Transfer of phloem-mobile substances from the host plants to the holoparasite Cuscuta sp.

PubMed

Birschwilks, Mandy; Haupt, Sophie; Hofius, Daniel; Neumann, Stefanie

2006-01-01

During the development of the haustorium, searching hyphae of the parasite and the host parenchyma cells are connected by plasmodesmata. Using transgenic tobacco plants expressing a GFP-labelled movement protein of the tobacco mosaic virus, it was demonstrated that the interspecific plasmodesmata are open. The transfer of substances in the phloem from host to the parasite is not selective. After simultaneous application of (3)H-sucrose and (14)C-labelled phloem-mobile amino acids, phytohormones, and xenobiotica to the host, corresponding percentages of the translocated compounds are found in the parasite. An open continuity between the host phloem and the Cuscuta phloem via the haustorium was demonstrated in CLSM pictures after application of the phloem-mobile fluorescent probes, carboxyfluorescein (CF) and hydroxypyrene trisulphonic acid (HPTS), to the host. Using a Cuscuta bridge (14)C-sucrose and the virus PVY(N) were transferred from one host plant to the another. The results of translocation experiments with labelled compounds, phloem-mobile dyes and the virus should be considered as unequivocal evidence for a symplastic transfer of phloem solutes between Cuscuta species and their compatible hosts.

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

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

Li, Zhou; Adams, Rachel M; Chourey, Karuna

2012-01-01

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

Application of Biosphere Compatibility Indicator for Assessment of the Effectiveness of Environmental Protection Methods

NASA Astrophysics Data System (ADS)

Bakaeva, N. V.; Vorobyov, S. A.; Chernyaeva, I. V.

2017-11-01

The article is devoted to the issue of using the biosphere compatibility indicator to assess the effectiveness of environmental protection methods. The indicator biosphere compatibility was proposed by the vice-president of RAASN (Russian Academy of Architecture and Building Sciences), Doctor of Technical Sciences, Professor V.I. Ilyichev. This indicator allows one to assess not only qualitatively but also quantitatively the degree of urban areas development from the standpoint of preserving the biosphere in urban ecosystems while performing the city’s main functions. The integral biosphere compatibility indicator allows us to assess not only the current ecological situation in the territory under consideration but also to plan the forecast of its changes for the new construction projects implementation or for the reconstruction of the existing ones. The biosphere compatibility indicator, which is a mathematical expression of the tripartite balance (technosphere, biosphere and population of this area), allows us to quantify the effectiveness degree of different methods for environment protection to choose the most effective one under these conditions.

Small-Molecule Organic Photovoltaic Modules Fabricated via Halogen-Free Solvent System with Roll-to-Roll Compatible Scalable Printing Method.

PubMed

Heo, Youn-Jung; Jung, Yen-Sook; Hwang, Kyeongil; Kim, Jueng-Eun; Yeo, Jun-Seok; Lee, Sehyun; Jeon, Ye-Jin; Lee, Donmin; Kim, Dong-Yu

2017-11-15

For the first time, the photovoltaic modules composed of small molecule were successfully fabricated by using roll-to-roll compatible printing techniques. In this study, blend films of small molecules, BTR and PC 71 BM were slot-die coated using a halogen-free solvent system. As a result, high efficiencies of 7.46% and 6.56% were achieved from time-consuming solvent vapor annealing (SVA) treatment and roll-to-roll compatible solvent additive approaches, respectively. After successful verification of our roll-to-roll compatible method on small-area devices, we further fabricated large-area photovoltaic modules with a total active area of 10 cm 2 , achieving a power conversion efficiency (PCE) of 4.83%. This demonstration of large-area photovoltaic modules through roll-to-roll compatible printing methods, even based on a halogen-free solvent, suggests the great potential for the industrial-scale production of organic solar cells (OSCs).

Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells

PubMed Central

Avilov, Sergiy; Magnus, Julie; Cusack, Stephen; Naffakh, Nadia

2016-01-01

Influenza viruses are a global health concern because of the permanent threat of novel emerging strains potentially capable of causing pandemics. Viral ribonucleoproteins (vRNPs) containing genomic RNA segments, nucleoprotein oligomers, and the viral polymerase, play a central role in the viral replication cycle. Our knowledge about critical events such as vRNP assembly and interactions with other viral and cellular proteins is poor and could be substantially improved by time lapse imaging of the infected cells. However, such studies are limited by the difficulty to achieve live-cell compatible labeling of active vRNPs. Previously we designed the first unimpaired recombinant influenza WSN-PB2-GFP11 virus allowing fluorescent labeling of the PB2 subunit of the viral polymerase (Avilov et al., J.Virol. 2012). Here, we simultaneously labeled the viral PB2 protein using the above-mentioned strategy, and virus-encoded progeny RNPs through spontaneous incorporation of transiently expressed NP-mCherry fusion proteins during RNP assembly in live infected cells. This dual labeling enabled us to visualize progeny vRNPs throughout the infection cycle and to characterize independently the mobility, oligomerization status and interactions of vRNP components in the nuclei of live infected cells. PMID:26978069

[Applications of mathematical statistics methods on compatibility researches of traditional Chinese medicines formulae].

PubMed

Mai, Lan-Yin; Li, Yi-Xuan; Chen, Yong; Xie, Zhen; Li, Jie; Zhong, Ming-Yu

2014-05-01

The compatibility of traditional Chinese medicines (TCMs) formulae containing enormous information, is a complex component system. Applications of mathematical statistics methods on the compatibility researches of traditional Chinese medicines formulae have great significance for promoting the modernization of traditional Chinese medicines and improving clinical efficacies and optimizations of formulae. As a tool for quantitative analysis, data inference and exploring inherent rules of substances, the mathematical statistics method can be used to reveal the working mechanisms of the compatibility of traditional Chinese medicines formulae in qualitatively and quantitatively. By reviewing studies based on the applications of mathematical statistics methods, this paper were summarized from perspective of dosages optimization, efficacies and changes of chemical components as well as the rules of incompatibility and contraindication of formulae, will provide the references for further studying and revealing the working mechanisms and the connotations of traditional Chinese medicines.

Fully automated synthesis of 4-[18F]fluorobenzylamine based on borohydride/NiCl2 reduction.

PubMed

Way, Jenilee; Wuest, Frank

2013-04-01

4-[(18)F]Fluorobenzylamine ([(18)F]FBA) is an important building block for the synthesis of (18)F-labeled compounds. Synthesis of [(18)F]FBA usually involves application of strong reducing agents like LiAlH4 which is challenging to handle in automated synthesis units (ASUs). Therefore, alternative methods for the preparation of [(18)F]FBA compatible with remotely-controlled syntheses in ASUs are needed. (18)F]FBA was prepared in a remotely-controlled synthesis unit (GE TRACERlab™ FX) based on Ni(II)-mediated borohydride exchange resin (BER) reduction of 4-[(18)F]fluorobenzonitrile ([(18)F]FBN). [(18)F]FBA was used for the synthesis of novel thiol-reactive prosthetic group 4-[(18)F]fluorobenzyl)maleimide [(18)F]FBM and Hsp90 inhibitor 17-(4-[(18)F]fluorobenzylamino)-17-demethoxy-geldanamycin [(18)F] GA. [(18)F]FBA could be prepared in high radiochemical yield greater than 80% (decay-corrected) within 60min. In a typical experiment, 7.4GBq of [(18)F]FBA could be obtained in high radiochemical purity of greater than 95% starting from 10GBq of cyclotron-produced n.c.a. [(18)F]fluoride. [(18)F]FBA was used for the preparation of 4-[(18)F]fluorobenzyl)maleimide as a novel prosthetic group for labeling of thiol groups as demonstrated with tripeptide glutathione. [(18)F]FBA was also used as building block for the syntheses of small molecules as exemplified by the preparation of Hsp90 inhibitor 17-(4-[(18)F]fluorobenzylamino)-17-demethoxy-geldanamycin. The described remotely-controlled synthesis of [(18)F]FBA will significantly improve the availability of [(18)F]FBA as an important and versatile building block for the development of novel (18)F-labeled compounds containing a fluorobenzylamine moiety. Copyright © 2013 Elsevier Inc. All rights reserved.

PyQuant: A Versatile Framework for Analysis of Quantitative Mass Spectrometry Data*

PubMed Central

Mitchell, Christopher J.; Kim, Min-Sik; Na, Chan Hyun; Pandey, Akhilesh

2016-01-01

Quantitative mass spectrometry data necessitates an analytical pipeline that captures the accuracy and comprehensiveness of the experiments. Currently, data analysis is often coupled to specific software packages, which restricts the analysis to a given workflow and precludes a more thorough characterization of the data by other complementary tools. To address this, we have developed PyQuant, a cross-platform mass spectrometry data quantification application that is compatible with existing frameworks and can be used as a stand-alone quantification tool. PyQuant supports most types of quantitative mass spectrometry data including SILAC, NeuCode, 15N, 13C, or 18O and chemical methods such as iTRAQ or TMT and provides the option of adding custom labeling strategies. In addition, PyQuant can perform specialized analyses such as quantifying isotopically labeled samples where the label has been metabolized into other amino acids and targeted quantification of selected ions independent of spectral assignment. PyQuant is capable of quantifying search results from popular proteomic frameworks such as MaxQuant, Proteome Discoverer, and the Trans-Proteomic Pipeline in addition to several standalone search engines. We have found that PyQuant routinely quantifies a greater proportion of spectral assignments, with increases ranging from 25–45% in this study. Finally, PyQuant is capable of complementing spectral assignments between replicates to quantify ions missed because of lack of MS/MS fragmentation or that were omitted because of issues such as spectra quality or false discovery rates. This results in an increase of biologically useful data available for interpretation. In summary, PyQuant is a flexible mass spectrometry data quantification platform that is capable of interfacing with a variety of existing formats and is highly customizable, which permits easy configuration for custom analysis. PMID:27231314

Preparation and evaluation of a 68Ga-labeled RGD-containing octapeptide for noninvasive imaging of angiogenesis: biodistribution in non-human primate

PubMed Central

Velikyan, Irina; Lindhe, Örjan

2018-01-01

Monitoring general disease marker such as angiogenesis may contribute to the development of personalized medicine and improve therapy outcome. Readily availability of positron emitter based imaging agents providing quantification would expand clinical positron emission tomography (PET) applications. Generator produced 68Ga provides PET images of high resolution and the half-life time frame is compatible with the pharmacokinetics of small peptides comprising arginine-glycine-aspartic acid (RGD) sequence specific to αvβ3 integrin receptors. The main objective of this study was to develop a method for 68Ga-labeling of RGD containing bicyclic octapeptide ([68Ga]Ga-DOTA-RGD) with high specific radioactivity and preclinically assess its imaging potential. DOTA-RGD was labeled using generator eluate preconcentration technique and microwave heating. The binding and organ distribution properties of [68Ga]Ga-DOTA-RGD were tested in vitro by autoradiography of frozen tumor sections, and in vivo in mice carrying a Lewis Lung carcinoma graft (LL2), and in non-human primate (NHP). Another peptide with aspartic acid-glycine-phenylalanine sequence was used as a negative control. The full 68Ga radioactivity eluted from two generators was quantitatively incorporated into 3-8 nanomoles of the peptide conjugates. The target binding specificity was confirmed by blocking experiments. The specific uptake in the LL2 mice model was observed in vivo and confirmed in the corresponding ex vivo biodistribution experiments. Increased accumulation of the radioactivity was detected in the wall of the uterus of the female NHP probably indicating neovascularization. [68Ga]Ga-DOTA-RGD demonstrated potential for the imaging of angiogenesis. PMID:29531858

Neural Network for Nanoscience Scanning Electron Microscope Image Recognition.

PubMed

Modarres, Mohammad Hadi; Aversa, Rossella; Cozzini, Stefano; Ciancio, Regina; Leto, Angelo; Brandino, Giuseppe Piero

2017-10-16

In this paper we applied transfer learning techniques for image recognition, automatic categorization, and labeling of nanoscience images obtained by scanning electron microscope (SEM). Roughly 20,000 SEM images were manually classified into 10 categories to form a labeled training set, which can be used as a reference set for future applications of deep learning enhanced algorithms in the nanoscience domain. The categories chosen spanned the range of 0-Dimensional (0D) objects such as particles, 1D nanowires and fibres, 2D films and coated surfaces, and 3D patterned surfaces such as pillars. The training set was used to retrain on the SEM dataset and to compare many convolutional neural network models (Inception-v3, Inception-v4, ResNet). We obtained compatible results by performing a feature extraction of the different models on the same dataset. We performed additional analysis of the classifier on a second test set to further investigate the results both on particular cases and from a statistical point of view. Our algorithm was able to successfully classify around 90% of a test dataset consisting of SEM images, while reduced accuracy was found in the case of images at the boundary between two categories or containing elements of multiple categories. In these cases, the image classification did not identify a predominant category with a high score. We used the statistical outcomes from testing to deploy a semi-automatic workflow able to classify and label images generated by the SEM. Finally, a separate training was performed to determine the volume fraction of coherently aligned nanowires in SEM images. The results were compared with what was obtained using the Local Gradient Orientation method. This example demonstrates the versatility and the potential of transfer learning to address specific tasks of interest in nanoscience applications.

Quantitative and Microscopic Assessment of Compatible and Incompatible Interactions between Chickpea Cultivars and Fusarium oxysporum f. sp. ciceris Races

PubMed Central

Jiménez-Fernández, Daniel; Landa, Blanca B.; Kang, Seogchan; Jiménez-Díaz, Rafael M.; Navas-Cortés, Juan A.

2013-01-01

Background Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris, a main threat to global chickpea production, is managed mainly by resistant cultivars whose efficiency is curtailed by Fusarium oxysporum f. sp. ciceris races. Methodology We characterized compatible and incompatible interactions by assessing the spatial-temporal pattern of infection and colonization of chickpea cvs. P-2245, JG-62 and WR-315 by Fusarium oxysporum f. sp. ciceris races 0 and 5 labeled with ZsGreen fluorescent protein using confocal laser scanning microscopy. Findings The two races colonized the host root surface in both interactions with preferential colonization of the root apex and subapical root zone. In compatible interactions, the pathogen grew intercellularly in the root cortex, reached the xylem, and progressed upwards in the stem xylem, being the rate and intensity of stem colonization directly related with the degree of compatibility among Fusarium oxysporum f. sp. ciceris races and chickpea cultivars. In incompatible interactions, race 0 invaded and colonized ‘JG-62’ xylem vessels of root and stem but in ‘WR-315’, it remained in the intercellular spaces of the root cortex failing to reach the xylem, whereas race 5 progressed up to the hypocotyl. However, all incompatible interactions were asymptomatic. Conclusions The differential patterns of colonization of chickpea cultivars by Fusarium oxysporum f. sp. ciceris races may be related to the operation of multiple resistance mechanisms. PMID:23613839

Compatibility: drugs and parenteral nutrition

PubMed Central

Miranda, Talita Muniz Maloni; Ferraresi, Andressa de Abreu

2016-01-01

ABSTRACT Objective Standardization and systematization of data to provide quick access to compatibility of leading injectable drugs used in hospitals for parenteral nutrition. Methods We selected 55 injectable drugs analyzed individually with two types of parenteral nutrition: 2-in-1 and 3-in-1. The following variables were considered: active ingredient, compatibility of drugs with the parenteral nutrition with or without lipids, and maximum drug concentration after dilution for the drugs compatible with parenteral nutrition. Drugs were classified as compatible, incompatible and untested. Results After analysis, relevant information to the product’s compatibility with parental nutrition was summarized in a table. Conclusion Systematization of compatibility data provided quick and easy access, and enabled standardizing pharmacists work. PMID:27074235

Multi-atlas label fusion using hybrid of discriminative and generative classifiers for segmentation of cardiac MR images.

PubMed

Sedai, Suman; Garnavi, Rahil; Roy, Pallab; Xi Liang

2015-08-01

Multi-atlas segmentation first registers each atlas image to the target image and transfers the label of atlas image to the coordinate system of the target image. The transferred labels are then combined, using a label fusion algorithm. In this paper, we propose a novel label fusion method which aggregates discriminative learning and generative modeling for segmentation of cardiac MR images. First, a probabilistic Random Forest classifier is trained as a discriminative model to obtain the prior probability of a label at the given voxel of the target image. Then, a probability distribution of image patches is modeled using Gaussian Mixture Model for each label, providing the likelihood of the voxel belonging to the label. The final label posterior is obtained by combining the classification score and the likelihood score under Bayesian rule. Comparative study performed on MICCAI 2013 SATA Segmentation Challenge demonstrates that our proposed hybrid label fusion algorithm is accurate than other five state-of-the-art label fusion methods. The proposed method obtains dice similarity coefficient of 0.94 and 0.92 in segmenting epicardium and endocardium respectively. Moreover, our label fusion method achieves more accurate segmentation results compared to four other label fusion methods.

A label distance maximum-based classifier for multi-label learning.

PubMed

Liu, Xiaoli; Bao, Hang; Zhao, Dazhe; Cao, Peng

2015-01-01

Multi-label classification is useful in many bioinformatics tasks such as gene function prediction and protein site localization. This paper presents an improved neural network algorithm, Max Label Distance Back Propagation Algorithm for Multi-Label Classification. The method was formulated by modifying the total error function of the standard BP by adding a penalty term, which was realized by maximizing the distance between the positive and negative labels. Extensive experiments were conducted to compare this method against state-of-the-art multi-label methods on three popular bioinformatic benchmark datasets. The results illustrated that this proposed method is more effective for bioinformatic multi-label classification compared to commonly used techniques.

A new real-time method for investigation of affinity properties and binding kinetics of magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Orlov, Alexey V.; Nikitin, Maxim P.; Bragina, Vera A.; Znoyko, Sergey L.; Zaikina, Marina N.; Ksenevich, Tatiana I.; Gorshkov, Boris G.; Nikitin, Petr I.

2015-04-01

A method for quantitative investigation of affinity constants of receptors immobilized on magnetic nanoparticles (MP) is developed based on spectral correlation interferometry (SCI). The SCI records with a picometer resolution the thickness changes of a layer of molecules or nanoparticles due to a biochemical reaction on a cover slip, averaged over the sensing area. The method is compatible with other types of sensing surfaces employed in biosensing. The measured values of kinetic association constants of magnetic nanoparticles are 4 orders of magnitude higher than those of molecular antibody association with antigen. The developed method also suggests highly sensitive detection of antigens in a wide dynamic range. The limit of detection of 92 pg/ml has been demonstrated for prostate-specific antigen (PSA) with 50-nm MP employed as labels, which produce 3-order amplification of the SCI signals. The calibration curve features high sensitivity (slope) of 3-fold signal raise per 10-fold increase of PSA concentration within 4-order dynamic range, which is an attractive compromise for precise quantitative and highly sensitive immunoassay. The proposed biosensing technique offers inexpensive disposable sensor chips of cover slips and represents an economically sound alternative to traditional immunoassays for disease diagnostics, detection of pathogens in food and environmental monitoring.

Electromagnetic Compatibility Design of the Computer Circuits

NASA Astrophysics Data System (ADS)

Zitai, Hong

2018-02-01

Computers and the Internet have gradually penetrated into every aspect of people’s daily work. But with the improvement of electronic equipment as well as electrical system, the electromagnetic environment becomes much more complex. Electromagnetic interference has become an important factor to hinder the normal operation of electronic equipment. In order to analyse the computer circuit compatible with the electromagnetic compatibility, this paper starts from the computer electromagnetic and the conception of electromagnetic compatibility. And then, through the analysis of the main circuit and system of computer electromagnetic compatibility problems, we can design the computer circuits in term of electromagnetic compatibility. Finally, the basic contents and methods of EMC test are expounded in order to ensure the electromagnetic compatibility of equipment.

Novel image processing method study for a label-free optical biosensor

NASA Astrophysics Data System (ADS)

Yang, Chenhao; Wei, Li'an; Yang, Rusong; Feng, Ying

2015-10-01

Optical biosensor is generally divided into labeled type and label-free type, the former mainly contains fluorescence labeled method and radioactive-labeled method, while fluorescence-labeled method is more mature in the application. The mainly image processing methods of fluorescent-labeled biosensor includes smooth filtering, artificial gridding and constant thresholding. Since some fluorescent molecules may influence the biological reaction, label-free methods have been the main developing direction of optical biosensors nowadays. The using of wider field of view and larger angle of incidence light path which could effectively improve the sensitivity of the label-free biosensor also brought more difficulties in image processing, comparing with the fluorescent-labeled biosensor. Otsu's method is widely applied in machine vision, etc, which choose the threshold to minimize the intraclass variance of the thresholded black and white pixels. It's capacity-constrained with the asymmetrical distribution of images as a global threshold segmentation. In order to solve the irregularity of light intensity on the transducer, we improved the algorithm. In this paper, we present a new image processing algorithm based on a reflectance modulation biosensor platform, which mainly comprises the design of sliding normalization algorithm for image rectification and utilizing the improved otsu's method for image segmentation, in order to implement automatic recognition of target areas. Finally we used adaptive gridding method extracting the target parameters for analysis. Those methods could improve the efficiency of image processing, reduce human intervention, enhance the reliability of experiments and laid the foundation for the realization of high throughput of label-free optical biosensors.

[Magnetic resonance compatibility research for coronary mental stents].

PubMed

Wang, Ying; Liu, Li; Wang, Shuo; Shang, Ruyao; Wang, Chunren

2015-01-01

The objective of this article is to research magnetic resonance compatibility for coronary mental stents, and to evaluate the magnetic resonance compatibility based on laboratory testing results. Coronary stents magnetic resonance compatibility test includes magnetically induced displacement force test, magnetically induced torque test, radio frequency induced heating and evaluation of MR image. By magnetic displacement force and torque values, temperature, and image distortion values to determine metal coronary stent demagnetization effect. The methods can be applied to test magnetic resonance compatibility for coronary mental stents and evaluate its demagnetization effect.

The performance evaluation model of mining project founded on the weight optimization entropy value method

NASA Astrophysics Data System (ADS)

Mao, Chao; Chen, Shou

2017-01-01

According to the traditional entropy value method still have low evaluation accuracy when evaluating the performance of mining projects, a performance evaluation model of mineral project founded on improved entropy is proposed. First establish a new weight assignment model founded on compatible matrix analysis of analytic hierarchy process (AHP) and entropy value method, when the compatibility matrix analysis to achieve consistency requirements, if it has differences between subjective weights and objective weights, moderately adjust both proportions, then on this basis, the fuzzy evaluation matrix for performance evaluation. The simulation experiments show that, compared with traditional entropy and compatible matrix analysis method, the proposed performance evaluation model of mining project based on improved entropy value method has higher accuracy assessment.

Compatibility of Segments of Thermoelectric Generators

NASA Technical Reports Server (NTRS)

Snyder, G. Jeffrey; Ursell, Tristan

2009-01-01

A method of calculating (usually for the purpose of maximizing) the power-conversion efficiency of a segmented thermoelectric generator is based on equations derived from the fundamental equations of thermoelectricity. Because it is directly traceable to first principles, the method provides physical explanations in addition to predictions of phenomena involved in segmentation. In comparison with the finite-element method used heretofore to predict (without being able to explain) the behavior of a segmented thermoelectric generator, this method is much simpler to implement in practice: in particular, the efficiency of a segmented thermoelectric generator can be estimated by evaluating equations using only hand-held calculator with this method. In addition, the method provides for determination of cascading ratios. The concept of cascading is illustrated in the figure and the definition of the cascading ratio is defined in the figure caption. An important aspect of the method is its approach to the issue of compatibility among segments, in combination with introduction of the concept of compatibility within a segment. Prior approaches involved the use of only averaged material properties. Two materials in direct contact could be examined for compatibility with each other, but there was no general framework for analysis of compatibility. The present method establishes such a framework. The mathematical derivation of the method begins with the definition of reduced efficiency of a thermoelectric generator as the ratio between (1) its thermal-to-electric power-conversion efficiency and (2) its Carnot efficiency (the maximum efficiency theoretically attainable, given its hot- and cold-side temperatures). The derivation involves calculation of the reduced efficiency of a model thermoelectric generator for which the hot-side temperature is only infinitesimally greater than the cold-side temperature. The derivation includes consideration of the ratio (u) between the electric current and heat-conduction power and leads to the concept of compatibility factor (s) for a given thermoelectric material, defined as the value of u that maximizes the reduced efficiency of the aforementioned model thermoelectric generator.

Effect of insecticides on mealybug destroyer (Coleoptera: Coccinellidae) and parasitoid Leptomastix dactylopii (Hymenoptera: Encyrtidae), natural enemies of citrus mealybug (Homoptera: Pseudococcidae).

PubMed

Cloyd, Raymond A; Dickinson, Amy

2006-10-01

In this study, we measured, under laboratory conditions, the direct and indirect effects of insecticides on mealybug destroyer, Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae), and parasitoid Leptomastix dactylopii Howard (Hymenoptera: Encyrtidae), natural enemies of citrus mealybug, Planococcus citri (Risso) (Homoptera: Pseudococcidae). The adult stages of both natural enemies were exposed to sprays of the insecticides buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran, and clothianidin at label-recommended rates to assess direct mortality after 24, 48, and 72 h, respectively. The effects of the insecticides on L. dactylopii parasitization rate and percentage of parasitoid emergence also were monitored using the label and 4x the recommended label rate. Dinotefuran was extremely detrimental to the adult parasitoid at the label rate with 100% mortality after 24 h. Buprofezin, pyriproxyfen, and flonicamid were not harmful to L. dactylopii when applied at the label rate. At 4x the recommended label rate, dinotefuran, acetamiprid, and clothianidin were all harmful to the parasitoid with 100% mortality 72 h after application. Both buprofezin and flonicamid were not toxic to L. dactylopii with 100% adult survival after 72 h. Pyriproxyfen and flonicamid, at both the label and 4x the recommended label rate, did not negatively affect L. dactylopii parasitization rate or percentage of parasitoid emergence. Acetamiprid, dinotefuran, and clothianidin were toxic to C. montrouzieri adults with 100% mortality after 48 h, whereas buprofezin, pyriproxyfen, and flonicamid demonstrated minimal (10-20% mortality after 48 h) harmful effects to the predator. Based on the results from our study, the indirect effects of the insect growth regulator (IGR) buprofezin were not decisive; however, the IGR pyriproxyfen and the insecticide flonicamid were not directly or indirectly harmful to the predator C. montrouzieri and parastioid L. dactylopii, indicating that these insecticides are compatible with both natural enemies when used together for control of citrus mealybug in greenhouses and conservatories.

Improved 68 Ga-labeling method using ethanol addition: Application to the α-helical peptide DOTA-FAMP.

PubMed

Hasegawa, Koki; Kawachi, Emi; Uehara, Yoshinari; Yoshida, Tsuyoshi; Imaizumi, Satoshi; Ogawa, Masahiro; Miura, Shin-Ichiro; Saku, Keijiro

2017-01-01

We examined the 68 Ga labeling of the α-helical peptide, DOTA-FAMP, and evaluated conformational changes during radiolabeling. 68 Ga-DOTA-FAMP is a positron emission tomography probe candidate for atherosclerotic plaques. The labeling yield achieved by Zhernosekov's method (using acetone for 68 Ga purification) was compared with that achieved by the original and 2 modified Mueller's methods (using NaCl solution). Modified method I involves desalting the 68 Ga prior to labeling, and modified method II involves the inclusion of ethanol in the labeling solution. The labeling yield using Zhernosekov's method was 62% ± 5.4%. In comparison, Mueller's original method gave 8.9% ± 1.7%. Modified method I gave a slight improvement of 32% ± 2.1%. Modified method II further increased the yield to 66% ± 3.4%. Conformational changes were determined by circular dichroism spectroscopy, revealing that these differences could be attributed to conformational changes. Heat treatment affects peptide conformation, which leads to aggregation and decreases the labeling yield. Mueller's method is simpler, but harsh conditions preclude its application to biomolecules. To suppress aggregation, we included a desalting process and added ethanol in the labeling solution. These changes significantly improved the labeling yield. Before use for imaging, conformational changes of biomolecules during radiolabeling should be evaluated by circular dichroism spectroscopy to ensure the homogeneity of the labeled product. Copyright © 2016 John Wiley & Sons, Ltd.

Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

PubMed Central

Volkmann, Gerrit; Liu, Xiang-Qin

2009-01-01

Background Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. Methodology A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. Conclusions We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups. PMID:20027230

An Interview with Matthew P. Greving, PhD. Interview by Vicki Glaser.

PubMed

Greving, Matthew P

2011-10-01

Matthew P. Greving is Chief Scientific Officer at Nextval Inc., a company founded in early 2010 that has developed a discovery platform called MassInsight™.. He received his PhD in Biochemistry from Arizona State University, and prior to that he spent nearly 7 years working as a software engineer. This experience in solving complex computational problems fueled his interest in developing technologies and algorithms related to acquisition and analysis of high-dimensional biochemical data. To address the existing problems associated with label-based microarray readouts, he beganwork on a technique for label-free mass spectrometry (MS) microarray readout compatible with both matrix-assisted laser/desorption ionization (MALDI) and matrix-free nanostructure initiator mass spectrometry (NIMS). This is the core of Nextval’s MassInsight technology, which utilizes picoliter noncontact deposition of high-density arrays on mass-readout substrates along with computational algorithms for high-dimensional data processingand reduction.

One-pot synthesis of fluorescent nitrogen-doped carbon dots with good biocompatibility for cell labeling.

PubMed

Zhang, Zhengwei; Yan, Kun; Yang, Qiulian; Liu, Yanhua; Yan, Zhengyu; Chen, Jianqiu

2017-12-01

Here we report an easy and economical hydrothermal carbonization approach to synthesize the fluorescent nitrogen-doped carbon dots (N-CDs) that was developed using citric acid and triethanolamine as the precursors. The synthesis conditions were optimized to obtain the N-CDs with superior fluorescence performances. The as-prepared N-CDs are monodispersed sphere nanoparticles with good water solubility, and exhibited strong fluorescence, favourable photostability and excitation wavelength-dependent behavior. Furthermore, the in vitro cytotoxicity and cellular labeling of N-CDs were investigated using the rat glomerular mesangial cells. The results showed the N-CDs have more inconspicuous cytotoxicity and better biosafety in comparison with ZnSe quantum dots, although both targeted the cells successfully. Considering their admirable photostability, low toxicity and good compatibility, the as-obtained N-CDs could have potential applications in biosensors, cellular imaging, and other fields. Copyright © 2017 John Wiley & Sons, Ltd.

PET/CT Based In Vivo Evaluation of 64Cu Labelled Nanodiscs in Tumor Bearing Mice.

PubMed

Huda, Pie; Binderup, Tina; Pedersen, Martin Cramer; Midtgaard, Søren Roi; Elema, Dennis Ringkjøbing; Kjær, Andreas; Jensen, Mikael; Arleth, Lise

2015-01-01

64Cu radiolabelled nanodiscs based on the 11 α-helix MSP1E3D1 protein and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipids were, for the first time, followed in vivo by positron emission tomography for evaluating the biodistribution of nanodiscs. A cancer tumor bearing mouse model was used for the investigations, and it was found that the approximately 13 nm nanodiscs, due to their size, permeate deeply into cancer tissue. This makes them promising candidates for both drug delivery purposes and as advanced imaging agents. For the radiolabelling, a simple approach for 64Cu radiolabelling of proteins via a chelating agent, DOTA, was developed. The reaction was performed at sufficiently mild conditions to be compatible with labelling of the protein part of a lipid-protein particle while fully conserving the particle structure including the amphipathic protein fold.

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

PubMed

Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

2018-02-13

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

Integration of genetically modified virus-like-particles with an optical resonator for selective bio-detection

NASA Astrophysics Data System (ADS)

Fan, X. Z.; Naves, L.; Siwak, N. P.; Brown, A.; Culver, J.; Ghodssi, R.

2015-05-01

A novel virus-like particle (TMV-VLP) receptor layer has been integrated with an optical microdisk resonator transducer for biosensing applications. This bioreceptor layer is functionalized with selective peptides that encode unique recognition affinities. Integration of bioreceptors with sensor platforms is very challenging due their very different compatibility regimes. The TMV-VLP nanoreceptor exhibits integration robustness, including the ability for self-assembly along with traditional top-down microfabrication processes. An optical microdisk resonator has been functionalized for antibody binding with this receptor, demonstrating resonant wavelength shifts of (Δλo) of 0.79 nm and 5.95 nm after primary antibody binding and enzyme-linked immunosorbent assay (ELISA), respectively, illustrating label-free sensing of this bonding event. This demonstration of label-free sensing with genetically engineered TMV-VLP shows the flexibility and utility of this receptor coating when considering integration with other existing transducer platforms.

Low temperature co-fired ceramic packaging of CMOS capacitive sensor chip towards cell viability monitoring.

PubMed

Halonen, Niina; Kilpijärvi, Joni; Sobocinski, Maciej; Datta-Chaudhuri, Timir; Hassinen, Antti; Prakash, Someshekar B; Möller, Peter; Abshire, Pamela; Kellokumpu, Sakari; Lloyd Spetz, Anita

2016-01-01

Cell viability monitoring is an important part of biosafety evaluation for the detection of toxic effects on cells caused by nanomaterials, preferably by label-free, noninvasive, fast, and cost effective methods. These requirements can be met by monitoring cell viability with a capacitance-sensing integrated circuit (IC) microchip. The capacitance provides a measurement of the surface attachment of adherent cells as an indication of their health status. However, the moist, warm, and corrosive biological environment requires reliable packaging of the sensor chip. In this work, a second generation of low temperature co-fired ceramic (LTCC) technology was combined with flip-chip bonding to provide a durable package compatible with cell culture. The LTCC-packaged sensor chip was integrated with a printed circuit board, data acquisition device, and measurement-controlling software. The packaged sensor chip functioned well in the presence of cell medium and cells, with output voltages depending on the medium above the capacitors. Moreover, the manufacturing of microfluidic channels in the LTCC package was demonstrated.

Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

PubMed

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-10-17

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Germanium Plasmon Enhanced Resonators for Label-Free Terahertz Protein Sensing

NASA Astrophysics Data System (ADS)

Bettenhausen, Maximilian; Römer, Friedhard; Witzigmann, Bernd; Flesch, Julia; Kurre, Rainer; Korneev, Sergej; Piehler, Jacob; You, Changjiang; Kazmierczak, Marcin; Guha, Subhajit; Capellini, Giovanni; Schröder, Thomas

2018-03-01

A Terahertz protein sensing concept based on subwavelength Ge resonators is presented. Ge bowtie resonators, compatible with CMOS fabrication technology, have been designed and characterized with a resonance frequency of 0.5 THz and calculated local intensity enhancement of 10.000. Selective biofunctionalization of Ge resonators on Si wafer was achieved in one step using lipoic acid-HaloTag ligand (LA-HTL) for biofunctionalization and passivation. The results lay the foundation for future investigation of protein tertiary structure and the dynamics of protein hydration shell in response to protein conformation changes.

Serotonin and serotoninergic neurons. A radioautographic and immunocytochemical study of the nucleus raphe dorsalis and nucleus dorsomedialis hypothalami.

PubMed

Arezki, F; Afailal, I; Bosler, O; Steinbusch, H W; Calas, A

1987-01-01

In an attempt to define cytophysiological criteria with which to establish whether or not a given neuron is serotoninergic, radioautography was combined with serotonin (5-HT) immunocytochemistry on the same sections from the nucleus raphe dorsalis (NRD) and/or nucleus dorsomedialis hypothalami (NDM) in rats subjected to intraventricular administrations of (3H)-5-HT or (3H)-dopamine (DA). All the (3H)-5-HT-accumulating neurons (cell bodies, dendrites and terminals) were found to be distinct from the (3H)-DA labeled ones and invariably immunostained for 5-HT in both regions studied. However, some immunoreactive neuronal elements within the area of tracer diffusion did not exhibit significant radioautographic labeling. In the NDM where 5-HT immunoreactive nerve cells could be detected only after intraventricular administration of 5-HT, these were found to be definitely distinct from the tyrosine hydroxylase immunoreactive and (3H)-DA labeled neurons of the dopaminergic periventricular-arcuate complex. After immunostaining for GAD at the electron microscopic level, (3H)-5-HT labeled nerve cells and terminals were not found to exhibit any significant immunoreactivity. Associations between (3H)-DA labeled and GAD immunoreactive processes with 5-HT immunoreactive or (3H)-5-HT-accumulating neurons, respectively, could also be observed in the NDM. When considered as a whole along with previous observations by other authors indicating a probable synthesis of 5-HT within NDM neurons, our data suggest that a given neuron can be classified as serotoninergic on the sole basis of its ability to selectively take up exogenous 5-HT under experimental conditions compatible with non interspecific labeling of catecholaminergic neurons. They also provide valuable information on the neurochemical environment and possible control of central serotoninergic neurons.

16 CFR 300.11 - Improper methods of labeling.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Improper methods of labeling. 300.11 Section 300.11 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.11 Improper methods of...

Building Systems Information Clearinghouse Special Report Number One: Manufacturers Compatibility Study.

ERIC Educational Resources Information Center

Boice, John R., Ed.

BSIC has selected data for inclusion, and a method of presentation that-- (1) provides preliminary data, in comparable form, about all relevant systems building products, (2) surveys within the limits imposed, the problems of compatibility between subsystem components and to identify components which are compatible with one another, (3) identifies…

Compatible poliomyelitis cases in India during 2000.

PubMed Central

Kohler, Kathryn A.; Hlady, W. Gary; Banerjee, Kaushik; Gupta, Dhananjoy; Francis, Paul; Durrani, Sunita; Zuber, Patrick L. F.; Sutter, Roland W.

2003-01-01

OBJECTIVE: To describe the characteristics of compatible poliomyelitis cases and to assess the programmatic implications of clusters of such cases in India. METHODS: We described the characteristics of compatible poliomyelitis cases, identified clusters of compatible cases (two or more in the same district or neighbouring districts within two months), and examined their relationship to wild poliovirus cases. FINDINGS: There were 362 compatible cases in 2000. The incidence of compatible cases was higher in districts with laboratory-confirmed poliomyelitis cases than in districts without laboratory-confirmed cases. Of 580 districts, 96 reported one compatible case and 72 reported two or more compatible cases. Among these 168 districts with at least one compatible case, 123 had internal or cross- border clusters of compatible cases. In 27 districts with clusters of compatible cases, no wild poliovirus was isolated either in the same district or in neighbouring districts. Three of these 27 districts presented laboratory-confirmed poliomyelitis cases during 2001. CONCLUSION: Most clusters of compatible cases occurred in districts identified as areas with continuing wild poliovirus transmission and where mopping-up vaccination campaigns were carried out. As certification nears, areas with compatible poliomyelitis cases should be investigated and deficiencies in surveillance should be corrected in order to ensure that certification is justified. PMID:12640469

Luminescent probes for optical in vivo imaging

NASA Astrophysics Data System (ADS)

Texier, Isabelle; Josserand, Veronique; Garanger, Elisabeth; Razkin, Jesus; Jin, Zhaohui; Dumy, Pascal; Favrot, Marie; Boturyn, Didier; Coll, Jean-Luc

2005-04-01

Going along with instrumental development for small animal fluorescence in vivo imaging, we are developing molecular fluorescent probes, especially for tumor targeting. Several criteria have to be taken into account for the optimization of the luminescent label. It should be adapted to the in vivo imaging optical conditions : red-shifted absorption and emission, limited overlap between absorption and emission for a good signal filtering, optimized luminescence quantum yield, limited photo-bleaching. Moreover, the whole probe should fulfill the biological requirements for in vivo labeling : adapted blood-time circulation, biological conditions compatibility, low toxicity. We here demonstrate the ability of the imaging fluorescence set-up developed in LETI to image the bio-distribution of molecular probes on short times after injection. Targeting with Cy5 labeled holo-transferrin of subcutaneous TS/Apc (angiogenic murine breast carcinoma model) or IGROV1 (human ovarian cancer) tumors was achieved. Differences in the kinetics of the protein uptake by the tumors were evidenced. IGROV1 internal metastatic nodes implanted in the peritoneal cavity could be detected in nude mice. However, targeted metastatic nodes in lung cancer could only be imaged after dissection of the mouse. These results validate our fluorescence imaging set-up and the use of Cy5 as a luminescent label. New fluorescent probes based on this dye and a molecular delivery template (the RAFT molecule) can thus be envisioned.

Document clustering methods, document cluster label disambiguation methods, document clustering apparatuses, and articles of manufacture

DOEpatents

Sanfilippo, Antonio [Richland, WA; Calapristi, Augustin J [West Richland, WA; Crow, Vernon L [Richland, WA; Hetzler, Elizabeth G [Kennewick, WA; Turner, Alan E [Kennewick, WA

2009-12-22

Document clustering methods, document cluster label disambiguation methods, document clustering apparatuses, and articles of manufacture are described. In one aspect, a document clustering method includes providing a document set comprising a plurality of documents, providing a cluster comprising a subset of the documents of the document set, using a plurality of terms of the documents, providing a cluster label indicative of subject matter content of the documents of the cluster, wherein the cluster label comprises a plurality of word senses, and selecting one of the word senses of the cluster label.

A review of traditional and current methods used to potentially reduce toxicity of Aconitum roots in Traditional Chinese Medicine.

PubMed

Liu, Shuai; Li, Fei; Li, Yan; Li, Weifei; Xu, Jinkai; Du, Hong

2017-07-31

Aconitum species are well-known for their medicinal value and high lethal toxicity in many Asian countries, notably China, India and Japan. The tubers are only used after processing in Traditional Chinese Medicine (TCM). They can be used safely and effectively with the methods of decoction, rational compatibility, and correct processing based on traditional experiences and new technologies. However, high toxicological risks still remain due to improper preparation and usage in China and other countries. Therefore, there is a need to clarify the methods of processing and compatibility to ensure their effectiveness and minimize the potential risks. The aim of this paper is to provide a review of traditional and current methods used to potentially reduce toxicity of Aconitum roots in TCM. The use of Aconitum has been investigated and the methods of processing and compatibility throughout history, including recent research, have been reviewed. Using of the methods of rational preparation, reasonable compatibility, and proper processing based on traditional experiences and new technologies, can enable Aconitum to be used safely and effectively. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Progressive multi-atlas label fusion by dictionary evolution.

PubMed

Song, Yantao; Wu, Guorong; Bahrami, Khosro; Sun, Quansen; Shen, Dinggang

2017-02-01

Accurate segmentation of anatomical structures in medical images is important in recent imaging based studies. In the past years, multi-atlas patch-based label fusion methods have achieved a great success in medical image segmentation. In these methods, the appearance of each input image patch is first represented by an atlas patch dictionary (in the image domain), and then the latent label of the input image patch is predicted by applying the estimated representation coefficients to the corresponding anatomical labels of the atlas patches in the atlas label dictionary (in the label domain). However, due to the generally large gap between the patch appearance in the image domain and the patch structure in the label domain, the estimated (patch) representation coefficients from the image domain may not be optimal for the final label fusion, thus reducing the labeling accuracy. To address this issue, we propose a novel label fusion framework to seek for the suitable label fusion weights by progressively constructing a dynamic dictionary in a layer-by-layer manner, where the intermediate dictionaries act as a sequence of guidance to steer the transition of (patch) representation coefficients from the image domain to the label domain. Our proposed multi-layer label fusion framework is flexible enough to be applied to the existing labeling methods for improving their label fusion performance, i.e., by extending their single-layer static dictionary to the multi-layer dynamic dictionary. The experimental results show that our proposed progressive label fusion method achieves more accurate hippocampal segmentation results for the ADNI dataset, compared to the counterpart methods using only the single-layer static dictionary. Copyright © 2016 Elsevier B.V. All rights reserved.

A new fluorogenic small molecule labeling tool for surface diffusion analysis and advanced fluorescence imaging of β-site amyloid precursor protein (APP)-cleaving enzyme 1 based on silicone rhodamine: SiR-BACE1.

PubMed

Karch, Sandra; Broichhagen, Johannes; Schneider, Julia; Böning, Daniel; Hartmann, Stephanie; Schmid, Benjamin; Tripal, Philipp; Palmisano, Ralf; Alzheimer, Christian; Johnsson, Kai; Huth, Tobias

2018-06-25

β-site APP-cleaving enzyme 1 (BACE1) is a major player in the pathogenesis of Alzheimer's disease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques either require genetic manipulation, use large antibodies, or are not compatible with live cell imaging. Fluorescent small molecules that specifically bind to the protein of interest can overcome these limitations. Herein, we introduce SiR-BACE1, a conjugate of the BACE1 inhibitor S-39 and SiR647, as a novel fluorogenic, tag-free, and antibody-free label for BACE1. We present its chemical development, characterize its photo-physical and pharmacologic properties, and evaluate its behavior in solution, in over-expression systems, and in native brain tissue. We demonstrate its applicability in confocal, stimulated emission depletion (STED), and dynamic single molecule microscopy. First functional studies with SiR-BACE1 on the surface mobility of BACE1 revealed a markedly confined diffusion pattern.

Reductive chemical release of N-glycans as 1-amino-alditols and subsequent 9-fluorenylmethyloxycarbonyl labeling for MS and LC/MS analysis.

PubMed

Wang, Chengjian; Qiang, Shan; Jin, Wanjun; Song, Xuezheng; Zhang, Ying; Huang, Linjuan; Wang, Zhongfu

2018-06-06

Glycoproteins play pivotal roles in a series of biological processes and their glycosylation patterns need to be structurally and functionally characterized. However, the lack of versatile methods to release N-glycans as functionalized forms has been undermining glycomics studies. Here a novel method is developed for dissociation of N-linked glycans from glycoproteins for analysis by MS and online LC/MS. This new method employs aqueous ammonia solution containing NaBH 3 CN as the reaction medium to release glycans from glycoproteins as 1-amino-alditol forms. The released glycans are conveniently labeled with 9-fluorenylmethyloxycarbonyl (Fmoc) and analyzed by ESI-MS and online LC/MS. Using the method, the neutral and acidic N-glycans were successfully released without peeling degradation of the core α-1,3-fucosylated structure or detectable de-N-acetylation, revealing its general applicability to various types of N-glycans. The Fmoc-derivatized N-glycans derived from chicken ovalbumin, Fagopyrum esculentum Moench Pollen and FBS were successfully analyzed by online LC/MS to distinguish isomers. The 1-amino-alditols were also permethylated to form quaternary ammonium cations at the reducing end, which enhance the MS sensitivity and are compatible with sequential multi-stage mass spectrometry (MS n ) fragmentation for glycan sequencing. The Fmoc-labeled N-glycans were further permethylated to produce methylated carbamates for determination of branches and linkages by sequential MS n fragmentation. N-Glycosylation represents one of the most common post-translational modification forms and plays pivotal roles in the structural and functional regulation of proteins in various biological activities, relating closely to human health and diseases. As a type of informational molecule, the N-glycans of glycoproteins participate directly in the molecular interactions between glycan epitopes and their corresponding protein receptors. Detailed structural and functional characterization of different types of N-glycans is essential for understanding the functional mechanisms of many biological activities and the pathologies of many diseases. Here we describe a simple, versatile method to indistinguishably release all types of N-glycans as functionalized forms without remarkable side reactions, enabling convenient, rapid analysis and preparation of released N-glycans from various complex biological samples. It is very valuable for studies on the complicated structure-function relationship of N-glycans, as well as for the search of N-glycan biomarkers of some major diseases and N-glycan related targets of some drugs. Copyright © 2018. Published by Elsevier B.V.

Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer

DOEpatents

Kwok, Pui-Yan; Chen, Xiangning

1999-01-01

A method for detecting the presence of a target nucleotide or sequence of nucleotides in a nucleic acid is disclosed. The method is comprised of forming an oligonucleotide labeled with two fluorophores on the nucleic acid target site. The doubly labeled oligonucleotide is formed by addition of a singly labeled dideoxynucleoside triphosphate to a singly labeled polynucleotide or by ligation of two singly labeled polynucleotides. Detection of fluorescence resonance energy transfer upon denaturation indicates the presence of the target. Kits are also provided. The method is particularly applicable to genotyping.

The interactive electrode localization utility: software for automatic sorting and labeling of intracranial subdural electrodes

PubMed Central

Tang, Wei; Peled, Noam; Vallejo, Deborah I.; Borzello, Mia; Dougherty, Darin D.; Eskandar, Emad N.; Widge, Alik S.; Cash, Sydney S.; Stufflebeam, Steven M.

2018-01-01

Purpose Existing methods for sorting, labeling, registering, and across-subject localization of electrodes in intracranial encephalography (iEEG) may involve laborious work requiring manual inspection of radiological images. Methods We describe a new open-source software package, the interactive electrode localization utility which presents a full pipeline for the registration, localization, and labeling of iEEG electrodes from CT and MR images. In addition, we describe a method to automatically sort and label electrodes from subdural grids of known geometry. Results We validated our software against manual inspection methods in twelve subjects undergoing iEEG for medically intractable epilepsy. Our algorithm for sorting and labeling performed correct identification on 96% of the electrodes. Conclusions The sorting and labeling methods we describe offer nearly perfect performance and the software package we have distributed may simplify the process of registering, sorting, labeling, and localizing subdural iEEG grid electrodes by manual inspection. PMID:27915398

Stem Cell Monitoring with a Direct or Indirect Labeling Method.

PubMed

Kim, Min Hwan; Lee, Yong Jin; Kang, Joo Hyun

2016-12-01

The molecular imaging techniques allow monitoring of the transplanted cells in the same individuals over time, from early localization to the survival, migration, and differentiation. Generally, there are two methods of stem cell labeling: direct and indirect labeling methods. The direct labeling method introduces a labeling agent into the cell, which is stably incorporated or attached to the cells prior to transplantation. Direct labeling of cells with radionuclides is a simple method with relatively fewer adverse events related to genetic responses. However, it can only allow short-term distribution of transplanted cells because of the decreasing imaging signal with radiodecay, according to the physical half-lives, or the signal becomes more diffuse with cell division and dispersion. The indirect labeling method is based on the expression of a reporter gene transduced into the cell before transplantation, which is then visualized upon the injection of an appropriate probe or substrate. In this review, various imaging strategies to monitor the survival and behavior change of transplanted stem cells are covered. Taking these new approaches together, the direct and indirect labeling methods may provide new insights on the roles of in vivo stem cell monitoring, from bench to bedside.

Ultra-small dye-doped silica nanoparticles via modified sol-gel technique.

PubMed

Riccò, R; Nizzero, S; Penna, E; Meneghello, A; Cretaio, E; Enrichi, F

2018-01-01

In modern biosensing and imaging, fluorescence-based methods constitute the most diffused approach to achieve optimal detection of analytes, both in solution and on the single-particle level. Despite the huge progresses made in recent decades in the development of plasmonic biosensors and label-free sensing techniques, fluorescent molecules remain the most commonly used contrast agents to date for commercial imaging and detection methods. However, they exhibit low stability, can be difficult to functionalise, and often result in a low signal-to-noise ratio. Thus, embedding fluorescent probes into robust and bio-compatible materials, such as silica nanoparticles, can substantially enhance the detection limit and dramatically increase the sensitivity. In this work, ultra-small fluorescent silica nanoparticles (NPs) for optical biosensing applications were doped with a fluorescent dye, using simple water-based sol-gel approaches based on the classical Stöber procedure. By systematically modulating reaction parameters, controllable size tuning of particle diameters as low as 10 nm was achieved. Particles morphology and optical response were evaluated showing a possible single-molecule behaviour, without employing microemulsion methods to achieve similar results. Graphical abstractWe report a simple, cheap, reliable protocol for the synthesis and systematic tuning of ultra-small (< 10 nm) dye-doped luminescent silica nanoparticles.

Vibrational spectroscopy for imaging single microbial cells in complex biological samples

DOE PAGES

Harrison, Jesse P.; Berry, David

2017-04-13

Here, vibrational spectroscopy is increasingly used for the rapid and non-destructive imaging of environmental and medical samples. Both Raman and Fourier-transform infrared (FT-IR) imaging have been applied to obtain detailed information on the chemical composition of biological materials, ranging from single microbial cells to tissues. Due to its compatibility with methods such as stable isotope labeling for the monitoring of cellular activities, vibrational spectroscopy also holds considerable power as a tool in microbial ecology. Chemical imaging of undisturbed biological systems (such as live cells in their native habitats) presents unique challenges due to the physical and chemical complexity of themore » samples, potential for spectral interference, and frequent need for real-time measurements. This Mini Review provides a critical synthesis of recent applications of Raman and FT-IR spectroscopy for characterizing complex biological samples, with a focus on developments in single-cell imaging. We also discuss how new spectroscopic methods could be used to overcome current limitations of singlecell analyses. Given the inherent complementarity of Raman and FT-IR spectroscopic methods, we discuss how combining these approaches could enable us to obtain new insights into biological activities either in situ or under conditions that simulate selected properties of the natural environment.« less

Vibrational spectroscopy for imaging single microbial cells in complex biological samples

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

Harrison, Jesse P.; Berry, David

Here, vibrational spectroscopy is increasingly used for the rapid and non-destructive imaging of environmental and medical samples. Both Raman and Fourier-transform infrared (FT-IR) imaging have been applied to obtain detailed information on the chemical composition of biological materials, ranging from single microbial cells to tissues. Due to its compatibility with methods such as stable isotope labeling for the monitoring of cellular activities, vibrational spectroscopy also holds considerable power as a tool in microbial ecology. Chemical imaging of undisturbed biological systems (such as live cells in their native habitats) presents unique challenges due to the physical and chemical complexity of themore » samples, potential for spectral interference, and frequent need for real-time measurements. This Mini Review provides a critical synthesis of recent applications of Raman and FT-IR spectroscopy for characterizing complex biological samples, with a focus on developments in single-cell imaging. We also discuss how new spectroscopic methods could be used to overcome current limitations of singlecell analyses. Given the inherent complementarity of Raman and FT-IR spectroscopic methods, we discuss how combining these approaches could enable us to obtain new insights into biological activities either in situ or under conditions that simulate selected properties of the natural environment.« less

[Compatible biomass models of natural spruce (Picea asperata)].

PubMed

Wang, Jin Chi; Deng, Hua Feng; Huang, Guo Sheng; Wang, Xue Jun; Zhang, Lu

2017-10-01

By using nonlinear measurement error method, the compatible tree volume and above ground biomass equations were established based on the volume and biomass data of 150 sampling trees of natural spruce (Picea asperata). Two approaches, controlling directly under total aboveground biomass and controlling jointly from level to level, were used to design the compatible system for the total aboveground biomass and the biomass of four components (stem, bark, branch and foliage), and the total ground biomass could be estimated independently or estimated simultaneously in the system. The results showed that the R 2 of the one variable and bivariate compatible tree volume and aboveground biomass equations were all above 0.85, and the maximum value reached 0.99. The prediction effect of the volume equations could be improved significantly when tree height was included as predictor, while it was not significant in biomass estimation. For the compatible biomass systems, the one variable model based on controlling jointly from level to level was better than the model using controlling directly under total above ground biomass, but the bivariate models of the two methods were similar. Comparing the imitative effects of the one variable and bivariate compatible biomass models, the results showed that the increase of explainable variables could significantly improve the fitness of branch and foliage biomass, but had little effect on other components. Besides, there was almost no difference between the two methods of estimation based on the comparison.

16 CFR 300.5 - Required label and method of affixing.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Required label and method of affixing. 300.5 Section 300.5 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.5 Required label and...

The origin of free brain malonate

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

Riley, K.M.; Dickson, A.C.; Koeppen, A.H.

Rat brain contains substantial concentrations of free malonate (192 nmol/g wet weight) but origin and biological importance of the dicarboxylic acid are poorly understood. A dietary source has been excluded. A recently described malonyl-CoA decarboxylase deficiency is associated with malonic aciduria and clinical manifestations, including mental retardation. In an effort to study the metabolic origin of free malonate, several labeled acetyl-CoA precursors were administered by intracerebral injection. (2-14C)pyruvate or (1,5-14C)citrate produced radioactive glutamate but failed to label malonate. In contrast, (1-14C)acetate, (2-14C)acetate, and (1-14C)butyrate were converted to labeled glutamate and malonate after the same route of administration. The intracerebral injectionmore » of (1-14C)-beta-alanine as a precursor of malonic semialdehyde and possibly free malonate did not give rise to radioactivity in the dicarboxylate. The labeling pattern of malonic acid is compatible with the reaction sequence: acetyl-CoA----malonyl-CoA----malonate. The final step is thought to occur by transfer of the CoA-group from malonyl-CoA to succinate and/or acetoacetate. Labeling of malonate from acetate is most effective at the age of 7 days when the net concentration of the dicarboxylic acid in rat brain is still very low. At this age, butyrate was a better precursor of malonate than acetate. It is proposed that fatty acid oxidation provides the acetyl-CoA which functions as the precursor of free brain malonate. Compartmentation of malonate biosynthesis is likely because the acetyl-CoA precursors citrate and pyruvate are ineffective.« less

In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism

PubMed Central

2012-01-01

Background The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes. Methods Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D3-Creatine (D3Cr) and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (CT1), L-arginine:glycine amidinotransferase (AGAT), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT) gene expression was assessed in the same cells by real time PCR. Results D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, AGAT, GAMT and SLC6A8 were expressed in both kind of cells. Conclusions Our results confirm that both neurons and astrocytes have the capability to synthesize and uptake Cr, and suggest that at least in vitro intracellular Cr can increase to a much greater extent through uptake than through de novo synthesis. Our results are compatible with the clinical observations that when the Cr transporter is defective, intracellular Cr is absent despite the brain should be able to synthesize it. Further research is needed to fully understand to what extent our results reflect the in vivo situation. PMID:22536786

Non-invasive monitoring of cytokine-based regenerative treatment of cartilage by hyperspectral unmixing (Conference Presentation)

NASA Astrophysics Data System (ADS)

Mahbub, Saabah B.; Succer, Peter; Gosnell, Martin E.; Anwaer, Ayad G.; Herbert, Benjamin; Vesey, Graham; Goldys, Ewa M.

2016-03-01

Extracting biochemical information from tissue autofluorescence is a promising approach to non-invasively monitor disease treatments at a cellular level, without using any external biomarkers. Our recently developed unsupervised hyperspectral unmixing by Dependent Component Analysis (DECA) provides robust and detailed metabolic information with proper account of intrinsic cellular heterogeneity. Moreover this method is compatible with established methods of fluorescent biomarker labelling. Recently adipose-derived stem cell (ADSC) - based therapies have been introduced for treating different diseases in animals and humans. ADSC have been shown promise in regenerative treatments for osteoarthritis and other bone and joint disorders. One of the mechanism of their action is their anti-inflammatory effects within osteoarthritic joints which aid the regeneration of cartilage. These therapeutic effects are known to be driven by secretions of different cytokines from the ADSCs. We have been using the hyperspectral unmixing techniques to study in-vitro the effects of ADSC-derived cytokine-rich secretions with the cartilage chip in both human and bovine samples. The study of metabolic effects of different cytokine treatment on different cartilage layers makes it possible to compare the merits of those treatments for repairing cartilage.

IMMUNOGLOBULIN ISOANTIGENS (ALLOTYPES) IN THE MOUSE

PubMed Central

Herzenberg, Leonard A.; Warner, Noel L.; Herzenberg, Leonore A.

1965-01-01

Eight antigens of 7S γ2-immunoglobulins controlled by alleles at a single locus Ig-1, have been identified in mice. This locus has previously been shown to determine antigenic specificities on the F fragments of 7S γ2a-globulins. The reactions of these antigens with various isoantisera have shown that the antigens all cross react with one another. New methods for the analysis of antigenic specificities of soluble proteins are presented in detail. A sensitive method for detecting in the order of 0.01 µg of these isoantigens has been developed, based on the quantitative inhibition of precipitation of I125-labeled antigen. Cross-reactions of the antigens were analysed in inhibition assays and the data is compatible with the existence of a minimum of eight antigenic specificities. Each of the antigens is composed of different combinations of these specificities, with only one antigen having a specificity not present in any other. Sixty-eight mouse strains have been tested with specific isoantisera, and on the basis of the results, have been placed into the eight allele groups. Evidence for close genetic linkage of the Ig-1 locus and 11 chromosome markers has been sought and not found. PMID:14270242

Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

PubMed

Hauser, Meghan; Wojcik, Michal; Kim, Doory; Mahmoudi, Morteza; Li, Wan; Xu, Ke

2017-06-14

Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

Assay for vitamin B12 absorption and method of making labeled vitamin B12

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

Anderson, Peter J; Dueker, Stephen; Miller, Joshua

2012-06-19

The invention provides methods for labeling vitamin B12 with .sup.14C, .sup.13C, tritium, and deuterium. When radioisotopes are used, the invention provides for methods of labeling B12 with high specific activity. The invention also provides labeled vitamin B12 compositions made in accordance with the invention.

Advanced techniques for determining long term compatibility of materials with propellants

NASA Technical Reports Server (NTRS)

Green, R. L.; Stebbins, J. P.; Smith, A. W.; Pullen, K. E.

1973-01-01

A method for the prediction of propellant-material compatibility for periods of time up to ten years is presented. Advanced sensitive measurement techniques used in the prediction method are described. These include: neutron activation analysis, radioactive tracer technique, and atomic absorption spectroscopy with a graphite tube furnace sampler. The results of laboratory tests performed to verify the prediction method are presented.

Revisiting GMOs: Are There Differences in European Consumers' Acceptance and Valuation for Cisgenically vs Transgenically Bred Rice?

PubMed

Delwaide, Anne-Cécile; Nalley, Lawton L; Dixon, Bruce L; Danforth, Diana M; Nayga, Rodolfo M; Van Loo, Ellen J; Verbeke, Wim

2015-01-01

Both cisgenesis and transgenesis are plant breeding techniques that can be used to introduce new genes into plant genomes. However, transgenesis uses gene(s) from a non-plant organism or from a donor plant that is sexually incompatible with the recipient plant while cisgenesis involves the introduction of gene(s) from a crossable--sexually compatible--plant. Traditional breeding techniques could possibly achieve the same results as those from cisgenesis, but would require a much larger timeframe. Cisgenesis allows plant breeders to enhance an existing cultivar more quickly and with little to no genetic drag. The current regulation in the European Union (EU) on genetically modified organisms (GMOs) treats cisgenic plants the same as transgenic plants and both are mandatorily labeled as GMOs. This study estimates European consumers' willingness-to-pay (WTP) for rice labeled as GM, cisgenic, with environmental benefits (which cisgenesis could provide), or any combination of these three attributes. Data were collected from 3,002 participants through an online survey administered in Belgium, France, the Netherlands, Spain and the United Kingdom in 2013. Censored regression models were used to model consumers' WTP in each country. Model estimates highlight significant differences in WTP across countries. In all five countries, consumers are willing-to-pay a premium to avoid purchasing rice labeled as GM. In all countries except Spain, consumers have a significantly higher WTP to avoid consuming rice labeled as GM compared to rice labeled as cisgenic, suggesting that inserting genes from the plant's own gene pool is more acceptable to consumers. Additionally, French consumers are willing-to-pay a premium for rice labeled as having environmental benefits compared to conventional rice. These findings suggest that not all GMOs are the same in consumers' eyes and thus, from a consumer preference perspective, the differences between transgenic and cisgenic products are recommended to be reflected in GMO labeling and trade policies.

A transversal approach for patch-based label fusion via matrix completion

PubMed Central

Sanroma, Gerard; Wu, Guorong; Gao, Yaozong; Thung, Kim-Han; Guo, Yanrong; Shen, Dinggang

2015-01-01

Recently, multi-atlas patch-based label fusion has received an increasing interest in the medical image segmentation field. After warping the anatomical labels from the atlas images to the target image by registration, label fusion is the key step to determine the latent label for each target image point. Two popular types of patch-based label fusion approaches are (1) reconstruction-based approaches that compute the target labels as a weighted average of atlas labels, where the weights are derived by reconstructing the target image patch using the atlas image patches; and (2) classification-based approaches that determine the target label as a mapping of the target image patch, where the mapping function is often learned using the atlas image patches and their corresponding labels. Both approaches have their advantages and limitations. In this paper, we propose a novel patch-based label fusion method to combine the above two types of approaches via matrix completion (and hence, we call it transversal). As we will show, our method overcomes the individual limitations of both reconstruction-based and classification-based approaches. Since the labeling confidences may vary across the target image points, we further propose a sequential labeling framework that first labels the highly confident points and then gradually labels more challenging points in an iterative manner, guided by the label information determined in the previous iterations. We demonstrate the performance of our novel label fusion method in segmenting the hippocampus in the ADNI dataset, subcortical and limbic structures in the LONI dataset, and mid-brain structures in the SATA dataset. We achieve more accurate segmentation results than both reconstruction-based and classification-based approaches. Our label fusion method is also ranked 1st in the online SATA Multi-Atlas Segmentation Challenge. PMID:26160394

Development of an automated modular system for the synthesis of [11C]acetate.

PubMed

Felicini, Chiara; Någren, Kjell; Berton, Andrea; Pascali, Giancarlo; Salvadori, Piero Alberto

2010-12-01

Carboxylation reactions offer a straightforward method for the synthesis of carbon-11 labelled carboxylic acids. Among these, the preparation of carbon-11 (C)-acetate is receiving increasing attention because of diagnostic applications in oncology in addition to its well-established use as a probe for myocardial oxidative metabolism. Although a number of dedicated modules are commercially available, the development of the synthesis on flexible platforms would be beneficial to widen the number of tracers, in particular for preclinical assessment and testing. In this study, the carboxylation reaction was implemented for the synthesis of sodium 1-[C]acetate after the classic route of carboxylation of methylmagnesium chloride by [C]carbon dioxide, followed by the acidic hydrolysis, purification and sterile filtration. This was performed using a commercially available kit of preassembled hardware units and fully compatible components of radiochemistry automation (VarioSystem). The system proved be to highly versatile and inexpensive and allowed a quick translation of the radiochemistry project into a working system even by less experienced personnel, because of predefined interfaces between electronic parts and operating software (preloaded on a laptop and included in the kit). The automatic module proved to be a simple and reliable system for the production of 1-[C]acetate that was prepared in 24 min (total synthesis time) with stable radiochemical yields (20% nondecay corrected) and high radiochemical purity (>97%). The module is used routinely to produce 1-[C]acetate for preclinical studies and is being implemented for the production of the labelled fatty acids.

Scleral fibroblast response to experimental glaucoma in mice

PubMed Central

Tezel, Gülgün; Cone-Kimball, Elizabeth; Steinhart, Matthew R.; Jefferys, Joan; Pease, Mary E.; Quigley, Harry A.

2016-01-01

Purpose To study the detailed cellular and molecular changes in the mouse sclera subjected to experimental glaucoma. Methods Three strains of mice underwent experimental bead-injection glaucoma and were euthanized at 3 days and 1, 3, and 6 weeks. Scleral protein expression was analyzed with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using 16O/18O labeling for quantification in 1- and 6-week tissues. Sclera protein samples were also analyzed with immunoblotting with specific antibodies to selected proteins. The proportion of proliferating scleral fibroblasts was quantified with Ki67 and 4’,6-diamidino-2-phenylindole (DAPI) labeling, and selected proteins were studied with immunohistochemistry. Results Proteomic analysis showed increases in molecules involved in integrin-linked kinase signaling and actin cytoskeleton signaling pathways at 1 and 6 weeks after experimental glaucoma. The peripapillary scleral region had more fibroblasts than equatorial sclera (p=0.001, n=217, multivariable regression models). There was a sixfold increase in proliferating fibroblasts in the experimental glaucoma sclera at 1 week and a threefold rise at 3 and 6 weeks (p=0.0005, univariate regression). Immunoblots confirmed increases for myosin, spectrin, and actinin at 1 week after glaucoma. Thrombospondin-1 (TSP-1), HINT1, vimentin, actinin, and α-smooth muscle actin were increased according to immunohistochemistry. Conclusions Scleral fibroblasts in experimental mouse glaucoma show increases in actin cytoskeleton and integrin-related signaling, increases in cell division, and features compatible with myofibroblast transition. PMID:26900327

'Isotopo' a database application for facile analysis and management of mass isotopomer data.

PubMed

Ahmed, Zeeshan; Zeeshan, Saman; Huber, Claudia; Hensel, Michael; Schomburg, Dietmar; Münch, Richard; Eylert, Eva; Eisenreich, Wolfgang; Dandekar, Thomas

2014-01-01

The composition of stable-isotope labelled isotopologues/isotopomers in metabolic products can be measured by mass spectrometry and supports the analysis of pathways and fluxes. As a prerequisite, the original mass spectra have to be processed, managed and stored to rapidly calculate, analyse and compare isotopomer enrichments to study, for instance, bacterial metabolism in infection. For such applications, we provide here the database application 'Isotopo'. This software package includes (i) a database to store and process isotopomer data, (ii) a parser to upload and translate different data formats for such data and (iii) an improved application to process and convert signal intensities from mass spectra of (13)C-labelled metabolites such as tertbutyldimethylsilyl-derivatives of amino acids. Relative mass intensities and isotopomer distributions are calculated applying a partial least square method with iterative refinement for high precision data. The data output includes formats such as graphs for overall enrichments in amino acids. The package is user-friendly for easy and robust data management of multiple experiments. The 'Isotopo' software is available at the following web link (section Download): http://spp1316.uni-wuerzburg.de/bioinformatics/isotopo/. The package contains three additional files: software executable setup (installer), one data set file (discussed in this article) and one excel file (which can be used to convert data from excel to '.iso' format). The 'Isotopo' software is compatible only with the Microsoft Windows operating system. http://spp1316.uni-wuerzburg.de/bioinformatics/isotopo/. © The Author(s) 2014. Published by Oxford University Press.

A small-scale open-label study of the treatment of canine flea allergy dermatitis with fluralaner.

PubMed

Fisara, Petr; Shipstone, Michael; von Berky, Andrew; von Berky, Janet

2015-12-01

Fluralaner is an isoxazoline systemic insecticide and acaricide that provides persistent flea-killing activity on dogs for 12 weeks. European and US field studies have shown that fluralaner treatment alleviates the signs of flea allergy dermatitis (FAD) in client-owned dogs. To assess the clinical response in FAD affected dogs over the 12-week period following a single oral fluralaner treatment. Twenty client-owned dogs were diagnosed with FAD on the basis of compatible clinical signs and a positive response in flea antigen tests, using intradermal and or serological methods. An open-label small-scale study with all dogs receiving a single oral fluralaner treatment. All enrolled dogs were diagnosed with FAD and then clinically monitored at 4-week intervals for 12 weeks. Twenty dogs completed the study. All dogs were flea-free at all post-treatment assessments except for one dog that had a single flea at the first post-enrollment assessment at 4 weeks. At the 4-week post-treatment assessment active FAD signs had resolved in all dogs; at 8 weeks post-treatment, two dogs showed mild signs. All clinical signs of FAD had resolved at the final assessment of 12 weeks after treatment. A single administration of fluralaner alleviated or resolved signs associated with FAD in all treated dogs over the recommended 12-week treatment period. © 2015 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of Intervet Australia Pty Ltd.

[Progress in stable isotope labeled quantitative proteomics methods].

PubMed

Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

2013-06-01

Quantitative proteomics is an important research field in post-genomics era. There are two strategies for proteome quantification: label-free methods and stable isotope labeling methods which have become the most important strategy for quantitative proteomics at present. In the past few years, a number of quantitative methods have been developed, which support the fast development in biology research. In this work, we discuss the progress in the stable isotope labeling methods for quantitative proteomics including relative and absolute quantitative proteomics, and then give our opinions on the outlook of proteome quantification methods.

The Catastrophe Revisited

PubMed Central

Ratner, Buddy D.

2007-01-01

The biomaterials community has been unable to accurately assign the term “blood compatible” to a biomaterial in spite of 50 years of intensive research on the subject. There is no clear consensus as to which materials are “blood compatible.” There are no standardized methods to assess blood compatibility. Since we use millions of devices in contact with blood each year, it is imperative we give serious thought to this intellectual catastrophe. In this perspective, I consider five hypotheses as to why progress has been slow in evolving a clear understanding of blood compatibility: Hypothesis 1 -- It is impossible to make a blood compatible material. Hypothesis 2 -- We do not understand the biology behind blood compatibility. Hypothesis 3 -- We do not understand how to test for or evaluate blood compatibility. Hypothesis 4 -- Certain materials of natural origin seem to show better blood compatibility but we do not know how to exploit this concept. Hypothesis 5 -- We now have better blood compatible materials but the regulatory and economic climate prevent adoption in clinical practice. PMID:17689608

MIPs in Aqueous Environments.

PubMed

Wan, Ying-chun; Ma, Hui-ting; Lu, Bin

2015-01-01

When organic solvent-compatible molecularly imprinted polymers (MIPs) are used in aqueous environment, how to reduce nonspecific binding is a major challenge. By modifying the binding solvents and introducing appropriate washing and elution steps, even relatively hydrophobic MIPs can gain optimal rebinding selectivity in aqueous conditions. Furthermore, water-compatible MIPs that can be used to treat aqueous samples directly have been prepared. The use of hydrophilic co-monomers, the controlled surface modification through controlled radical polymerization, and the new interfacial molecular imprinting methods are different strategies to prepare water-compatible MIPs. By combining MIPs with other techniques, both organic solvent-compatible and water-compatible MIPs can display better functional performances in aqueous conditions. Intensive studies on MIPs in aqueous conditions can provide new MIPs with much-improved compatibilities that will lead to more interesting applications in biomedicine and biotechnology.

Constraining Genome-Scale Models to Represent the Bow Tie Structure of Metabolism for 13C Metabolic Flux Analysis

PubMed Central

Ando, David; Singh, Jahnavi; Keasling, Jay D.; García Martín, Héctor

2018-01-01

Determination of internal metabolic fluxes is crucial for fundamental and applied biology because they map how carbon and electrons flow through metabolism to enable cell function. 13C Metabolic Flux Analysis (13C MFA) and Two-Scale 13C Metabolic Flux Analysis (2S-13C MFA) are two techniques used to determine such fluxes. Both operate on the simplifying approximation that metabolic flux from peripheral metabolism into central “core” carbon metabolism is minimal, and can be omitted when modeling isotopic labeling in core metabolism. The validity of this “two-scale” or “bow tie” approximation is supported both by the ability to accurately model experimental isotopic labeling data, and by experimentally verified metabolic engineering predictions using these methods. However, the boundaries of core metabolism that satisfy this approximation can vary across species, and across cell culture conditions. Here, we present a set of algorithms that (1) systematically calculate flux bounds for any specified “core” of a genome-scale model so as to satisfy the bow tie approximation and (2) automatically identify an updated set of core reactions that can satisfy this approximation more efficiently. First, we leverage linear programming to simultaneously identify the lowest fluxes from peripheral metabolism into core metabolism compatible with the observed growth rate and extracellular metabolite exchange fluxes. Second, we use Simulated Annealing to identify an updated set of core reactions that allow for a minimum of fluxes into core metabolism to satisfy these experimental constraints. Together, these methods accelerate and automate the identification of a biologically reasonable set of core reactions for use with 13C MFA or 2S-13C MFA, as well as provide for a substantially lower set of flux bounds for fluxes into the core as compared with previous methods. We provide an open source Python implementation of these algorithms at https://github.com/JBEI/limitfluxtocore. PMID:29300340

Comparison of tissue processing methods for microvascular visualization in axolotls.

PubMed

Montoro, Rodrigo; Dickie, Renee

2017-01-01

The vascular system, the pipeline for oxygen and nutrient delivery to tissues, is essential for vertebrate development, growth, injury repair, and regeneration. With their capacity to regenerate entire appendages throughout their lifespan, axolotls are an unparalleled model for vertebrate regeneration, but they lack many of the molecular tools that facilitate vascular imaging in other animal models. The determination of vascular metrics requires high quality image data for the discrimination of vessels from background tissue. Quantification of the vasculature using perfused, cleared specimens is well-established in mammalian systems, but has not been widely employed in amphibians. The objective of this study was to optimize tissue preparation methods for the visualization of the microvascular network in axolotls, providing a basis for the quantification of regenerative angiogenesis. To accomplish this aim, we performed intracardiac perfusion of pigment-based contrast agents and evaluated aqueous and non-aqueous clearing techniques. The methods were verified by comparing the quality of the vascular images and the observable vascular density across treatment groups. Simple and inexpensive, these tissue processing techniques will be of use in studies assessing vascular growth and remodeling within the context of regeneration. Advantages of this method include: •Higher contrast of the vasculature within the 3D context of the surrounding tissue •Enhanced detection of microvasculature facilitating vascular quantification •Compatibility with other labeling techniques.

Method for inducing hypothermia

DOEpatents

Becker, Lance B.; Hoek, Terry Vanden; Kasza, Kenneth E.

2003-04-15

Systems for phase-change particulate slurry cooling equipment and methods to induce hypothermia in a patient through internal and external cooling are provided. Subcutaneous, intravascular, intraperitoneal, gastrointestinal, and lung methods of cooling are carried out using saline ice slurries or other phase-change slurries compatible with human tissue. Perfluorocarbon slurries or other slurry types compatible with human tissue are used for pulmonary cooling. And traditional external cooling methods are improved by utilizing phase-change slurry materials in cooling caps and torso blankets.

Method for inducing hypothermia

DOEpatents

Becker, Lance B [Chicago, IL; Hoek, Terry Vanden [Chicago, IL; Kasza, Kenneth E [Palos Park, IL

2008-09-09

Systems for phase-change particulate slurry cooling equipment and methods to induce hypothermia in a patient through internal and external cooling are provided. Subcutaneous, intravascular, intraperitoneal, gastrointestinal, and lung methods of cooling are carried out using saline ice slurries or other phase-change slurries compatible with human tissue. Perfluorocarbon slurries or other slurry types compatible with human tissue are used for pulmonary cooling. And traditional external cooling methods are improved by utilizing phase-change slurry materials in cooling caps and torso blankets.

Method for inducing hypothermia

DOEpatents

Becker, Lance B.; Hoek, Terry Vanden; Kasza, Kenneth E.

2005-11-08

Systems for phase-change particulate slurry cooling equipment and methods to induce hypothermia in a patient through internal and external cooling are provided. Subcutaneous, intravascular, intraperitoneal, gastrointestinal, and lung methods of cooling are carried out using saline ice slurries or other phase-change slurries compatible with human tissue. Perfluorocarbon slurries or other slurry types compatible with human tissue are used for pulmonary cooling. And traditional external cooling methods are improved by utilizing phase-change slurry materials in cooling caps and torso blankets.

Waveguide-type optical circuits for recognition of optical 8QAM-coded label

NASA Astrophysics Data System (ADS)

Surenkhorol, Tumendemberel; Kishikawa, Hiroki; Goto, Nobuo; Gonchigsumlaa, Khishigjargal

2017-10-01

Optical signal processing is expected to be applied in network nodes. In photonic routers, label recognition is one of the important functions. We have studied different kinds of label recognition methods so far for on-off keying, binary phase-shift keying, quadrature phase-shift keying, and 16 quadrature amplitude modulation-coded labels. We propose a method based on waveguide circuits to recognize an optical eight quadrature amplitude modulation (8QAM)-coded label by simple passive optical signal processing. The recognition of the proposed method is theoretically analyzed and numerically simulated by the finite difference beam propagation method. The noise tolerance is discussed, and bit-error rate against optical signal-to-noise ratio is evaluated. The scalability of the proposed method is also discussed theoretically for two-symbol length 8QAM-coded labels.

Progressive Label Fusion Framework for Multi-atlas Segmentation by Dictionary Evolution

PubMed Central

Song, Yantao; Wu, Guorong; Sun, Quansen; Bahrami, Khosro; Li, Chunming; Shen, Dinggang

2015-01-01

Accurate segmentation of anatomical structures in medical images is very important in neuroscience studies. Recently, multi-atlas patch-based label fusion methods have achieved many successes, which generally represent each target patch from an atlas patch dictionary in the image domain and then predict the latent label by directly applying the estimated representation coefficients in the label domain. However, due to the large gap between these two domains, the estimated representation coefficients in the image domain may not stay optimal for the label fusion. To overcome this dilemma, we propose a novel label fusion framework to make the weighting coefficients eventually to be optimal for the label fusion by progressively constructing a dynamic dictionary in a layer-by-layer manner, where a sequence of intermediate patch dictionaries gradually encode the transition from the patch representation coefficients in image domain to the optimal weights for label fusion. Our proposed framework is general to augment the label fusion performance of the current state-of-the-art methods. In our experiments, we apply our proposed method to hippocampus segmentation on ADNI dataset and achieve more accurate labeling results, compared to the counterpart methods with single-layer dictionary. PMID:26942233

Progressive Label Fusion Framework for Multi-atlas Segmentation by Dictionary Evolution.

PubMed

Song, Yantao; Wu, Guorong; Sun, Quansen; Bahrami, Khosro; Li, Chunming; Shen, Dinggang

2015-10-01

Accurate segmentation of anatomical structures in medical images is very important in neuroscience studies. Recently, multi-atlas patch-based label fusion methods have achieved many successes, which generally represent each target patch from an atlas patch dictionary in the image domain and then predict the latent label by directly applying the estimated representation coefficients in the label domain. However, due to the large gap between these two domains, the estimated representation coefficients in the image domain may not stay optimal for the label fusion. To overcome this dilemma, we propose a novel label fusion framework to make the weighting coefficients eventually to be optimal for the label fusion by progressively constructing a dynamic dictionary in a layer-by-layer manner, where a sequence of intermediate patch dictionaries gradually encode the transition from the patch representation coefficients in image domain to the optimal weights for label fusion. Our proposed framework is general to augment the label fusion performance of the current state-of-the-art methods. In our experiments, we apply our proposed method to hippocampus segmentation on ADNI dataset and achieve more accurate labeling results, compared to the counterpart methods with single-layer dictionary.

Development and application of a robust N-glycan profiling method for heightened characterization of monoclonal antibodies and related glycoproteins.

PubMed

Shang, Tanya Q; Saati, Andrew; Toler, Kelly N; Mo, Jianming; Li, Heyi; Matlosz, Tonya; Lin, Xi; Schenk, Jennifer; Ng, Chee-Keng; Duffy, Toni; Porter, Thomas J; Rouse, Jason C

2014-07-01

A highly robust hydrophilic interaction liquid chromatography (HILIC) method that involves both fluorescence and mass spectrometric detection was developed for profiling and characterizing enzymatically released and 2-aminobenzamide (2-AB)-derivatized mAb N-glycans. Online HILIC/mass spectrometry (MS) with a quadrupole time-of-flight mass spectrometer provides accurate mass identifications of the separated, 2-AB-labeled N-glycans. The method features a high-resolution, low-shedding HILIC column with acetonitrile and water-based mobile phases containing trifluoroacetic acid (TFA) as a modifier. This column and solvent system ensures the combination of robust chromatographic performance and full compatibility and sensitivity with online MS in addition to the baseline separation of all typical mAb N-glycans. The use of TFA provided distinct advantages over conventional ammonium formate as a mobile phase additive, such as, optimal elution order for sialylated N-glycans, reproducible chromatographic profiles, and matching total ion current chromatograms, as well as minimal signal splitting, analyte adduction, and fragmentation during HILIC/MS, maximizing sensitivity for trace-level species. The robustness and selectivity of HILIC for N-glycan analyses allowed for method qualification. The method is suitable for bioprocess development activities, heightened characterization, and clinical drug substance release. Application of this HILIC/MS method to the detailed characterization of a marketed therapeutic mAb, Rituxan(®), is described. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

A simple and fast detection method for bovine milk residues in foods: a 2-site monoclonal antibody immunochromatography assay.

PubMed

Xuli, Wu; Weiyi, He; Ji, Kunmei; Wenpu, Wan; Dongsheng, Hu; Hui, Wu; Xinpin, Luo; Zhigang, Liu

2013-03-01

The ingredient declaration on food labels assumes paramount importance in the protection of food-allergic consumers. China has not implemented Food allergen labeling. A gold immunochromatography assay (GICA) was developed using 2 monoclonal antibodies (mAb) against the milk allergen β-lactoglobulin in this study. The GICA was specific for pure milk samples with a sensitivity of 0.2 ng/mL. Milk protein traces extracted from 110 food products were detected by this method. The labels of 106 were confirmed by our GICA method: 57 food samples originally labeled as containing milk were positive for β-lactoglobulin and 49 food samples labeled as not containing milk were negative for β-lactoglobulin. However, 3 food samples falsely labeled as containing milk were found to contain no β-lactoglobulin whereas 1 food sample labeled as not containing milk actually contained β-lactoglobulin. First, these negatives could be because of the addition of a casein fraction. Second, some countries demand that food manufacturers label all ingredients derived from milk as "containing milk" even though the ingredients contain no detectable milk protein by any method. Our GICA method could thus provide a fast and simple method for semiquantitatation of β-lactoglobulin in foods. The present method provides a fast, simple, semiquantitative method for the determination of milk allergens in foods. © 2013 Institute of Food Technologists®

Microfluidic Cell Culture Device

NASA Technical Reports Server (NTRS)

Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

2014-01-01

Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

Microfabricated capillary electrophoresis chip and method for simultaneously detecting multiple redox labels

DOEpatents

Mathies, Richard A.; Singhal, Pankaj; Xie, Jin; Glazer, Alexander N.

2002-01-01

This invention relates to a microfabricated capillary electrophoresis chip for detecting multiple redox-active labels simultaneously using a matrix coding scheme and to a method of selectively labeling analytes for simultaneous electrochemical detection of multiple label-analyte conjugates after electrophoretic or chromatographic separation.

Direct coordinate-free derivation of the compatibility equation for finite strains

NASA Astrophysics Data System (ADS)

Ryzhak, E. I.

2014-07-01

The compatibility equation for the Cauchy-Green tensor field (squared tensor of pure extensionwith respect to the reference configuration) is directly derived from the well-known relation expressing this tensor via the vector field determining the mapping (transformation) of the reference configuration into the actual one. The derivation is based on the use of the apparatus of coordinatefree tensor calculus and does not apply any notions and relations of Riemannian geometry at all. The method is illustrated by deriving the well-known compatibility equation for small strains. It is shown that when the obtained compatibility equation for finite strains is linearized, it becomes the compatibility equation for small strains which indirectly confirms its correctness.

Learning classification models with soft-label information.

PubMed

Nguyen, Quang; Valizadegan, Hamed; Hauskrecht, Milos

2014-01-01

Learning of classification models in medicine often relies on data labeled by a human expert. Since labeling of clinical data may be time-consuming, finding ways of alleviating the labeling costs is critical for our ability to automatically learn such models. In this paper we propose a new machine learning approach that is able to learn improved binary classification models more efficiently by refining the binary class information in the training phase with soft labels that reflect how strongly the human expert feels about the original class labels. Two types of methods that can learn improved binary classification models from soft labels are proposed. The first relies on probabilistic/numeric labels, the other on ordinal categorical labels. We study and demonstrate the benefits of these methods for learning an alerting model for heparin induced thrombocytopenia. The experiments are conducted on the data of 377 patient instances labeled by three different human experts. The methods are compared using the area under the receiver operating characteristic curve (AUC) score. Our AUC results show that the new approach is capable of learning classification models more efficiently compared to traditional learning methods. The improvement in AUC is most remarkable when the number of examples we learn from is small. A new classification learning framework that lets us learn from auxiliary soft-label information provided by a human expert is a promising new direction for learning classification models from expert labels, reducing the time and cost needed to label data.

Direct, rapid, and label-free detection of enzyme-substrate interactions in physiological buffers using CMOS-compatible nanoribbon sensors.

PubMed

Mu, Luye; Droujinine, Ilia A; Rajan, Nitin K; Sawtelle, Sonya D; Reed, Mark A

2014-09-10

We demonstrate the versatility of Al2O3-passivated Si nanowire devices ("nanoribbons") in the analysis of enzyme-substrate interactions via the monitoring of pH change. Our approach is shown to be effective through the detection of urea in phosphate buffered saline (PBS), and penicillinase in PBS and urine, at limits of detection of <200 μM and 0.02 units/mL, respectively. The ability to extract accurate enzyme kinetics and the Michaelis-Menten constant (Km) from the acetylcholine-acetylcholinesterase reaction is also demonstrated.

Nonlocal atlas-guided multi-channel forest learning for human brain labeling

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

Ma, Guangkai; Gao, Yaozong; Wu, Guorong

Purpose: It is important for many quantitative brain studies to label meaningful anatomical regions in MR brain images. However, due to high complexity of brain structures and ambiguous boundaries between different anatomical regions, the anatomical labeling of MR brain images is still quite a challenging task. In many existing label fusion methods, appearance information is widely used. However, since local anatomy in the human brain is often complex, the appearance information alone is limited in characterizing each image point, especially for identifying the same anatomical structure across different subjects. Recent progress in computer vision suggests that the context features canmore » be very useful in identifying an object from a complex scene. In light of this, the authors propose a novel learning-based label fusion method by using both low-level appearance features (computed from the target image) and high-level context features (computed from warped atlases or tentative labeling maps of the target image). Methods: In particular, the authors employ a multi-channel random forest to learn the nonlinear relationship between these hybrid features and target labels (i.e., corresponding to certain anatomical structures). Specifically, at each of the iterations, the random forest will output tentative labeling maps of the target image, from which the authors compute spatial label context features and then use in combination with original appearance features of the target image to refine the labeling. Moreover, to accommodate the high inter-subject variations, the authors further extend their learning-based label fusion to a multi-atlas scenario, i.e., they train a random forest for each atlas and then obtain the final labeling result according to the consensus of results from all atlases. Results: The authors have comprehensively evaluated their method on both public LONI-LBPA40 and IXI datasets. To quantitatively evaluate the labeling accuracy, the authors use the dice similarity coefficient to measure the overlap degree. Their method achieves average overlaps of 82.56% on 54 regions of interest (ROIs) and 79.78% on 80 ROIs, respectively, which significantly outperform the baseline method (random forests), with the average overlaps of 72.48% on 54 ROIs and 72.09% on 80 ROIs, respectively. Conclusions: The proposed methods have achieved the highest labeling accuracy, compared to several state-of-the-art methods in the literature.« less

Nonlocal atlas-guided multi-channel forest learning for human brain labeling

PubMed Central

Ma, Guangkai; Gao, Yaozong; Wu, Guorong; Wu, Ligang; Shen, Dinggang

2016-01-01

Purpose: It is important for many quantitative brain studies to label meaningful anatomical regions in MR brain images. However, due to high complexity of brain structures and ambiguous boundaries between different anatomical regions, the anatomical labeling of MR brain images is still quite a challenging task. In many existing label fusion methods, appearance information is widely used. However, since local anatomy in the human brain is often complex, the appearance information alone is limited in characterizing each image point, especially for identifying the same anatomical structure across different subjects. Recent progress in computer vision suggests that the context features can be very useful in identifying an object from a complex scene. In light of this, the authors propose a novel learning-based label fusion method by using both low-level appearance features (computed from the target image) and high-level context features (computed from warped atlases or tentative labeling maps of the target image). Methods: In particular, the authors employ a multi-channel random forest to learn the nonlinear relationship between these hybrid features and target labels (i.e., corresponding to certain anatomical structures). Specifically, at each of the iterations, the random forest will output tentative labeling maps of the target image, from which the authors compute spatial label context features and then use in combination with original appearance features of the target image to refine the labeling. Moreover, to accommodate the high inter-subject variations, the authors further extend their learning-based label fusion to a multi-atlas scenario, i.e., they train a random forest for each atlas and then obtain the final labeling result according to the consensus of results from all atlases. Results: The authors have comprehensively evaluated their method on both public LONI_LBPA40 and IXI datasets. To quantitatively evaluate the labeling accuracy, the authors use the dice similarity coefficient to measure the overlap degree. Their method achieves average overlaps of 82.56% on 54 regions of interest (ROIs) and 79.78% on 80 ROIs, respectively, which significantly outperform the baseline method (random forests), with the average overlaps of 72.48% on 54 ROIs and 72.09% on 80 ROIs, respectively. Conclusions: The proposed methods have achieved the highest labeling accuracy, compared to several state-of-the-art methods in the literature. PMID:26843260

Static and dynamic behaviour of nonlocal elastic bar using integral strain-based and peridynamic models

NASA Astrophysics Data System (ADS)

Challamel, Noël

2018-04-01

The static and dynamic behaviour of a nonlocal bar of finite length is studied in this paper. The nonlocal integral models considered in this paper are strain-based and relative displacement-based nonlocal models; the latter one is also labelled as a peridynamic model. For infinite media, and for sufficiently smooth displacement fields, both integral nonlocal models can be equivalent, assuming some kernel correspondence rules. For infinite media (or finite media with extended reflection rules), it is also shown that Eringen's differential model can be reformulated into a consistent strain-based integral nonlocal model with exponential kernel, or into a relative displacement-based integral nonlocal model with a modified exponential kernel. A finite bar in uniform tension is considered as a paradigmatic static case. The strain-based nonlocal behaviour of this bar in tension is analyzed for different kernels available in the literature. It is shown that the kernel has to fulfil some normalization and end compatibility conditions in order to preserve the uniform strain field associated with this homogeneous stress state. Such a kernel can be built by combining a local and a nonlocal strain measure with compatible boundary conditions, or by extending the domain outside its finite size while preserving some kinematic compatibility conditions. The same results are shown for the nonlocal peridynamic bar where a homogeneous strain field is also analytically obtained in the elastic bar for consistent compatible kinematic boundary conditions at the vicinity of the end conditions. The results are extended to the vibration of a fixed-fixed finite bar where the natural frequencies are calculated for both the strain-based and the peridynamic models.

Compatibility of the Parasitoid Wasp Spalangia endius (Hymenoptera: Pteromalidae) and Insecticides against Musca domestica (Diptera: Muscidae) as Evaluated by a New Index.

PubMed

Burgess, Edwin R; King, B H

2015-06-01

Various insecticides for the control of the house fly Musca domestica L. were tested for compatibility with a biological control agent, the pupal parasitoid Spalangia endius Walker. Bioassays used the mode in which each organism was expected to be harmed by the insecticides, a surface contact bioassay for S. endius and a feeding bioassay for M. domestica. A Pesticide Compatibility Index (PCI) was created that allows comparison of LC50 values when the mode of exposure to a pesticide differs. First LC50 values were converted into units of prescribed dosages (LPR=LC50-to-prescribed dosage ratio). This study used dosages from labels of granular baits. PCI is the ratio of LPRbiological control agent to LPRpest. For these PCI values, order of compatibility with S. endius was spinosad>thiamethoxam>inotefuran>methomyl>imidacloprid. That spinosad was better than imidacloprid or methomyl, both for parasitoid survival and for killing flies, is consistent with conclusions from the LC50 values. Permethrin and nitenpyram were also tested, but their PCIs were not calculated. Permethrin is prescribed as a contact insecticide against flies rather than being consumed as a bait, and nitenpyram has not been formulated as a fly insecticide. Compared with the other insecticides in terms of LC50 values, permethrin was moderately toxic to S. endius but one of the most toxic for M. domestica, whereas nitenpyram was least toxic for both S. endius and the flies. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Label-free functional nucleic acid sensors for detecting target agents

DOEpatents

Lu, Yi; Xiang, Yu

2015-01-13

A general methodology to design label-free fluorescent functional nucleic acid sensors using a vacant site approach and an abasic site approach is described. In one example, a method for designing label-free fluorescent functional nucleic acid sensors (e.g., those that include a DNAzyme, aptamer or aptazyme) that have a tunable dynamic range through the introduction of an abasic site (e.g., dSpacer) or a vacant site into the functional nucleic acids. Also provided is a general method for designing label-free fluorescent aptamer sensors based on the regulation of malachite green (MG) fluorescence. A general method for designing label-free fluorescent catalytic and molecular beacons (CAMBs) is also provided. The methods demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes. Sensors and methods of using the disclosed sensors are also provided.

A general soft label based linear discriminant analysis for semi-supervised dimensionality reduction.

PubMed

Zhao, Mingbo; Zhang, Zhao; Chow, Tommy W S; Li, Bing

2014-07-01

Dealing with high-dimensional data has always been a major problem in research of pattern recognition and machine learning, and Linear Discriminant Analysis (LDA) is one of the most popular methods for dimension reduction. However, it only uses labeled samples while neglecting unlabeled samples, which are abundant and can be easily obtained in the real world. In this paper, we propose a new dimension reduction method, called "SL-LDA", by using unlabeled samples to enhance the performance of LDA. The new method first propagates label information from the labeled set to the unlabeled set via a label propagation process, where the predicted labels of unlabeled samples, called "soft labels", can be obtained. It then incorporates the soft labels into the construction of scatter matrixes to find a transformed matrix for dimension reduction. In this way, the proposed method can preserve more discriminative information, which is preferable when solving the classification problem. We further propose an efficient approach for solving SL-LDA under a least squares framework, and a flexible method of SL-LDA (FSL-LDA) to better cope with datasets sampled from a nonlinear manifold. Extensive simulations are carried out on several datasets, and the results show the effectiveness of the proposed method. Copyright © 2014 Elsevier Ltd. All rights reserved.

Performance limitations of label-free sensors in molecular diagnosis using complex samples

NASA Astrophysics Data System (ADS)

Varma, Manoj

2016-03-01

Label-free biosensors promised a paradigm involving direct detection of biomarkers from complex samples such as serum without requiring multistep sample processing typical of labelled methods such as ELISA or immunofluorescence assays. Label-free sensors have witnessed decades of development with a veritable zoo of techniques available today exploiting a multitude of physical effects. It is appropriate now to critically assess whether label-free technologies have succeeded in delivering their promise with respect to diagnostic applications, particularly, ambitious goals such as early cancer detection using serum biomarkers, which require low limits of detection (LoD). Comparison of nearly 120 limits of detection (LoD) values reported by labelled and label-free sensing approaches over a wide range of detection techniques and target molecules in serum revealed that labeled techniques achieve 2-3 orders of magnitude better LoDs. Data from experiments where labelled and label-free assays were performed simultaneously using the same assay parameters also confirm that the LoD achieved by labelled techniques is 2 to 3 orders of magnitude better than that by label-free techniques. Furthermore, label-free techniques required significant signal amplification, for e.g. using nanoparticle conjugated secondary antibodies, to achieve LoDs comparable to labelled methods substantially deviating from the original "direct detection" paradigm. This finding has important implications on the practical limits of applying label-free detection methods for molecular diagnosis.

Evaluation of Four Methods for Predicting Carbon Stocks of Korean Pine Plantations in Heilongjiang Province, China

PubMed Central

Gao, Huilin; Dong, Lihu; Li, Fengri; Zhang, Lianjun

2015-01-01

A total of 89 trees of Korean pine (Pinus koraiensis) were destructively sampled from the plantations in Heilongjiang Province, P.R. China. The sample trees were measured and calculated for the biomass and carbon stocks of tree components (i.e., stem, branch, foliage and root). Both compatible biomass and carbon stock models were developed with the total biomass and total carbon stocks as the constraints, respectively. Four methods were used to evaluate the carbon stocks of tree components. The first method predicted carbon stocks directly by the compatible carbon stocks models (Method 1). The other three methods indirectly predicted the carbon stocks in two steps: (1) estimating the biomass by the compatible biomass models, and (2) multiplying the estimated biomass by three different carbon conversion factors (i.e., carbon conversion factor 0.5 (Method 2), average carbon concentration of the sample trees (Method 3), and average carbon concentration of each tree component (Method 4)). The prediction errors of estimating the carbon stocks were compared and tested for the differences between the four methods. The results showed that the compatible biomass and carbon models with tree diameter (D) as the sole independent variable performed well so that Method 1 was the best method for predicting the carbon stocks of tree components and total. There were significant differences among the four methods for the carbon stock of stem. Method 2 produced the largest error, especially for stem and total. Methods 3 and Method 4 were slightly worse than Method 1, but the differences were not statistically significant. In practice, the indirect method using the mean carbon concentration of individual trees was sufficient to obtain accurate carbon stocks estimation if carbon stocks models are not available. PMID:26659257

Optimization and quality assessment of the post-digestion 18O labeling based on urea for protein denaturation by HPLC/ESI-TOF mass spectrometry.

PubMed

Wang, Hongbin; Hu, Gaofei; Zhang, Yongqian; Yuan, Zheng; Zhao, Xuan; Zhu, Yong; Cai, De; Li, Yujuan; Xiao, Shengyuan; Deng, Yulin

2010-07-15

The post-digestion (18)O labeling method decouples protein digestion and peptide labeling. This method allows labeling conditions to be optimized separately and increases labeling efficiency. A common method for protein denaturation in proteomics is the use of urea. Though some previous studies have used urea-based protein denaturation before post-digestion (18)O labeling, the optimal (18)O labeling conditions in this case have not been yet reported. Present study investigated the effects of urea concentration and pH on the labeling efficiency and obtained an optimized protocol. It was demonstrated that urea inhibited (18)O incorporation depending on concentration. However, a urea concentration between 1 and 2M had minimal effects on labeling. It was also demonstrated that the use of FA to quench the digestion reaction severely affected the labeling efficiency. This study revealed the reason why previous studies gave different optimal pH for labeling. They neglect the effects of different digestion conditions on the labeling conditions. Excellent labeling quality was obtained at the optimized conditions using urea 1-2 M and pH 4.5, 98.4+/-1.9% for a standard protein mixture and 97.2+/-6.2% for a complex biological sample. For a 1:1 mixture analysis of the (16)O- and (18)O-labeled peptides from the same protein sample, the average abundance ratios reached 1.05+/-0.31, demonstrating a good quantitation quality at the optimized conditions. This work will benefit other researchers who pair urea-based protein denaturation with a post-digestion (18)O labeling method. 2010 Elsevier B.V. All rights reserved.

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

PubMed

Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

2018-04-19

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

A dual-colored bio-marker made of doped ZnO nanocrystals

NASA Astrophysics Data System (ADS)

Wu, Y. L.; Fu, S.; Tok, A. I. Y.; Zeng, X. T.; Lim, C. S.; Kwek, L. C.; Boey, F. C. Y.

2008-08-01

Bio-compatible ZnO nanocrystals doped with Co, Cu and Ni cations, surface capped with two types of aminosilanes and titania are synthesized by a soft chemical process. Due to the small particle size (2-5 nm), surface functional groups and the high photoluminescence emissions at the UV and blue-violet wavelength ranges, bio-imaging on human osteosarcoma (Mg-63) cells and histiocytic lymphoma U-937 monocyte cells showed blue emission at the nucleus and bright turquoise emission at the cytoplasm simultaneously. This is the first report on dual-color bio-images labeled by one semiconductor nanocrystal colloidal solution. Bright green emission was detected on mung bean seedlings labeled by all the synthesized ZnO nanocrystals. Cytotoxicity tests showed that the aminosilanes capped nanoparticles are non-toxic. Quantum yields of the nanocrystals varied from 79% to 95%. The results showed the potential of the pure ZnO and Co-doped ZnO nanocrystals for live imaging of both human cells and plant systems.

Technical Note: PLASTIMATCH MABS, an open source tool for automatic image segmentation

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

Zaffino, Paolo; Spadea, Maria Francesca

Purpose: Multiatlas based segmentation is largely used in many clinical and research applications. Due to its good performances, it has recently been included in some commercial platforms for radiotherapy planning and surgery guidance. Anyway, to date, a software with no restrictions about the anatomical district and image modality is still missing. In this paper we introduce PLASTIMATCH MABS, an open source software that can be used with any image modality for automatic segmentation. Methods: PLASTIMATCH MABS workflow consists of two main parts: (1) an offline phase, where optimal registration and voting parameters are tuned and (2) an online phase, wheremore » a new patient is labeled from scratch by using the same parameters as identified in the former phase. Several registration strategies, as well as different voting criteria can be selected. A flexible atlas selection scheme is also available. To prove the effectiveness of the proposed software across anatomical districts and image modalities, it was tested on two very different scenarios: head and neck (H&N) CT segmentation for radiotherapy application, and magnetic resonance image brain labeling for neuroscience investigation. Results: For the neurological study, minimum dice was equal to 0.76 (investigated structures: left and right caudate, putamen, thalamus, and hippocampus). For head and neck case, minimum dice was 0.42 for the most challenging structures (optic nerves and submandibular glands) and 0.62 for the other ones (mandible, brainstem, and parotid glands). Time required to obtain the labels was compatible with a real clinical workflow (35 and 120 min). Conclusions: The proposed software fills a gap in the multiatlas based segmentation field, since all currently available tools (both for commercial and for research purposes) are restricted to a well specified application. Furthermore, it can be adopted as a platform for exploring MABS parameters and as a reference implementation for comparing against other segmentation algorithms.« less

PyQuant: A Versatile Framework for Analysis of Quantitative Mass Spectrometry Data.

PubMed

Mitchell, Christopher J; Kim, Min-Sik; Na, Chan Hyun; Pandey, Akhilesh

2016-08-01

Quantitative mass spectrometry data necessitates an analytical pipeline that captures the accuracy and comprehensiveness of the experiments. Currently, data analysis is often coupled to specific software packages, which restricts the analysis to a given workflow and precludes a more thorough characterization of the data by other complementary tools. To address this, we have developed PyQuant, a cross-platform mass spectrometry data quantification application that is compatible with existing frameworks and can be used as a stand-alone quantification tool. PyQuant supports most types of quantitative mass spectrometry data including SILAC, NeuCode, (15)N, (13)C, or (18)O and chemical methods such as iTRAQ or TMT and provides the option of adding custom labeling strategies. In addition, PyQuant can perform specialized analyses such as quantifying isotopically labeled samples where the label has been metabolized into other amino acids and targeted quantification of selected ions independent of spectral assignment. PyQuant is capable of quantifying search results from popular proteomic frameworks such as MaxQuant, Proteome Discoverer, and the Trans-Proteomic Pipeline in addition to several standalone search engines. We have found that PyQuant routinely quantifies a greater proportion of spectral assignments, with increases ranging from 25-45% in this study. Finally, PyQuant is capable of complementing spectral assignments between replicates to quantify ions missed because of lack of MS/MS fragmentation or that were omitted because of issues such as spectra quality or false discovery rates. This results in an increase of biologically useful data available for interpretation. In summary, PyQuant is a flexible mass spectrometry data quantification platform that is capable of interfacing with a variety of existing formats and is highly customizable, which permits easy configuration for custom analysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Physics of a rapid CD4 lymphocyte count with colloidal gold.

PubMed

Hansen, P; Barry, D; Restell, A; Sylvia, D; Magnin, O; Dombkowski, D; Preffer, F

2012-03-01

The inherent surface charges and small diameters that confer colloidal stability to gold particle conjugates (immunogold) are detrimental to rapid cell surface labeling and distinct cluster definition in flow cytometric light scatter assays. Although the inherent immunogold surface charge prevents self aggregation when stored in liquid suspension, it also slows binding to cells to timeframes of hours and inhibits cell surface coverage. Although the small diameter of immunogold particles prevents settling when in liquid suspension, small particles have small light scattering cross sections and weak light scatter signals. We report a new, small particle lyophilized immunogold reagent that maintains activity after 42°C storage for a year and can be rapidly dissolved into stable liquid suspension for use in labelling cells with larger particle aggregates that have enhanced scattering cross section. Labeling requires less than 1 min at 20°C, which is ∼30 times faster than customary fluorescent antibody labeling. The labeling step involves neutralizing the surface charge of immunogold and creating specifically bound aggregates of gold on the cell surface. This process provides distinct side-scatter cluster separation with blue laser light at 488 nm, which is further improved by using red laser light at 640 nm. Similar comparisons using LED light sources showed less improvement with red light, thereby indicating that coherent light scatter is of significance in enhancing side-scatter cluster separation. The physical principles elucidated here for this technique are compatible with most flow cytometers; however, future studies of its clinical efficacy should be of primary interest in point-of-care applications where robust reagents and rapid results are important. Copyright © 2011 International Society for Advancement of Cytometry.

Efficient Multi-Atlas Registration using an Intermediate Template Image

PubMed Central

Dewey, Blake E.; Carass, Aaron; Blitz, Ari M.; Prince, Jerry L.

2017-01-01

Multi-atlas label fusion is an accurate but time-consuming method of labeling the human brain. Using an intermediate image as a registration target can allow researchers to reduce time constraints by storing the deformations required of the atlas images. In this paper, we investigate the effect of registration through an intermediate template image on multi-atlas label fusion and propose a novel registration technique to counteract the negative effects of through-template registration. We show that overall computation time can be decreased dramatically with minimal impact on final label accuracy and time can be exchanged for improved results in a predictable manner. We see almost complete recovery of Dice similarity over a simple through-template registration using the corrected method and still maintain a 3–4 times speed increase. Further, we evaluate the effectiveness of this method on brains of patients with normal-pressure hydrocephalus, where abnormal brain shape presents labeling difficulties, specifically the ventricular labels. Our correction method creates substantially better ventricular labeling than traditional methods and maintains the speed increase seen in healthy subjects. PMID:28943702

Efficient multi-atlas registration using an intermediate template image

NASA Astrophysics Data System (ADS)

Dewey, Blake E.; Carass, Aaron; Blitz, Ari M.; Prince, Jerry L.

2017-03-01

Multi-atlas label fusion is an accurate but time-consuming method of labeling the human brain. Using an intermediate image as a registration target can allow researchers to reduce time constraints by storing the deformations required of the atlas images. In this paper, we investigate the effect of registration through an intermediate template image on multi-atlas label fusion and propose a novel registration technique to counteract the negative effects of through-template registration. We show that overall computation time can be decreased dramatically with minimal impact on final label accuracy and time can be exchanged for improved results in a predictable manner. We see almost complete recovery of Dice similarity over a simple through-template registration using the corrected method and still maintain a 3-4 times speed increase. Further, we evaluate the effectiveness of this method on brains of patients with normal-pressure hydrocephalus, where abnormal brain shape presents labeling difficulties, specifically the ventricular labels. Our correction method creates substantially better ventricular labeling than traditional methods and maintains the speed increase seen in healthy subjects.

Label Information Guided Graph Construction for Semi-Supervised Learning.

PubMed

Zhuang, Liansheng; Zhou, Zihan; Gao, Shenghua; Yin, Jingwen; Lin, Zhouchen; Ma, Yi

2017-09-01

In the literature, most existing graph-based semi-supervised learning methods only use the label information of observed samples in the label propagation stage, while ignoring such valuable information when learning the graph. In this paper, we argue that it is beneficial to consider the label information in the graph learning stage. Specifically, by enforcing the weight of edges between labeled samples of different classes to be zero, we explicitly incorporate the label information into the state-of-the-art graph learning methods, such as the low-rank representation (LRR), and propose a novel semi-supervised graph learning method called semi-supervised low-rank representation. This results in a convex optimization problem with linear constraints, which can be solved by the linearized alternating direction method. Though we take LRR as an example, our proposed method is in fact very general and can be applied to any self-representation graph learning methods. Experiment results on both synthetic and real data sets demonstrate that the proposed graph learning method can better capture the global geometric structure of the data, and therefore is more effective for semi-supervised learning tasks.

Compatibility of pedigree-based and marker-based relationship matrices for single-step genetic evaluation.

PubMed

Christensen, Ole F

2012-12-03

Single-step methods provide a coherent and conceptually simple approach to incorporate genomic information into genetic evaluations. An issue with single-step methods is compatibility between the marker-based relationship matrix for genotyped animals and the pedigree-based relationship matrix. Therefore, it is necessary to adjust the marker-based relationship matrix to the pedigree-based relationship matrix. Moreover, with data from routine evaluations, this adjustment should in principle be based on both observed marker genotypes and observed phenotypes, but until now this has been overlooked. In this paper, I propose a new method to address this issue by 1) adjusting the pedigree-based relationship matrix to be compatible with the marker-based relationship matrix instead of the reverse and 2) extending the single-step genetic evaluation using a joint likelihood of observed phenotypes and observed marker genotypes. The performance of this method is then evaluated using two simulated datasets. The method derived here is a single-step method in which the marker-based relationship matrix is constructed assuming all allele frequencies equal to 0.5 and the pedigree-based relationship matrix is constructed using the unusual assumption that animals in the base population are related and inbred with a relationship coefficient γ and an inbreeding coefficient γ / 2. Taken together, this γ parameter and a parameter that scales the marker-based relationship matrix can handle the issue of compatibility between marker-based and pedigree-based relationship matrices. The full log-likelihood function used for parameter inference contains two terms. The first term is the REML-log-likelihood for the phenotypes conditional on the observed marker genotypes, whereas the second term is the log-likelihood for the observed marker genotypes. Analyses of the two simulated datasets with this new method showed that 1) the parameters involved in adjusting marker-based and pedigree-based relationship matrices can depend on both observed phenotypes and observed marker genotypes and 2) a strong association between these two parameters exists. Finally, this method performed at least as well as a method based on adjusting the marker-based relationship matrix. Using the full log-likelihood and adjusting the pedigree-based relationship matrix to be compatible with the marker-based relationship matrix provides a new and interesting approach to handle the issue of compatibility between the two matrices in single-step genetic evaluation.

Method And Apparatus For Production Of Bi-213 From The Activity Ac-225 Source

DOEpatents

Egorov, Oleg B.; O'Hara, Matthew J.

2005-12-06

A method and apparatus for isolating and purifying a .sup.213 Bi radioactive isotope from an .sup.225 Ac source using a primary column and a primary sorbent which preferentially retains .sup.225 Ac over .sup.213 Bi when exposed to a compatible solvent in combination with a secondary column having a secondary sorbent which retains .sup.213 Bi when exposed to a mixture of the compatible solvent and .sup.213 Bi. A "compatible solvent" is a solvent which will preferentially remove .sup.213 Bi radioactive isotopes from a primary sorbent without removing .sup.225 Ac radioactive isotopes, and then allow .sup.213 Bi radioactive isotopes removed from the primary sorbent to be retained on a secondary sorbent, without having to dilute or otherwise chemically or physically modify the compatible solvent in between exposure to the primary and secondary sorbents.

Bacterial toxicity/compatibility of platinum nanospheres, nanocuboids and nanoflowers

PubMed Central

Gopal, Judy; Hasan, Nazim; Manikandan, M.; Wu, Hui-Fen

2013-01-01

For the first time, we have investigated the bacterial toxicity or compatibility properties of Pt nanoparticles (NPs) with different sizes (P1, P2, P3, P4 and P5). The bacterio-toxic or compatible properties of these five different sized Pt NPs with the clinical pathogen, Pseudomonas aeruginosa were explored by many analytical methods such as the conventional plate count method, matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), fluorescence microscopy and fluorescence sensoring techniques. The results revealed that the 1–3 nm sized (P1 and P2) Pt NPs showed bacterio-toxic properties while the 4–21 nm (P3, P4 and P5) Pt NPs exhibited bacterio-compatible properties. This is the first study which reports the bacterial toxicity of Pt NPs. The information released from this study is significantly important to future clinical, medical, biological and biomedical applications of Pt NPs. PMID:23405274

Hydrocarbon-fuel/combustion-chamber-liner materials compatibility

NASA Technical Reports Server (NTRS)

Gage, Mark L.

1990-01-01

Results of material compatibility experiments using hydrocarbon fuels in contact with copper-based combustion chamber liner materials are presented. Mil-Spec RP-1, n- dodecane, propane, and methane fuels were tested in contact with OFHC, NASA-Z, and ZrCu coppers. Two distinct test methods were employed. Static tests, in which copper coupons were exposed to fuel for long durations at constant temperature and pressure, provided compatibility data in a precisely controlled environment. Dynamic tests, using the Aerojet Carbothermal Test Facility, provided fuel and copper compatibility data under realistic booster engine service conditions. Tests were conducted using very pure grades of each fuel and fuels to which a contaminant, e.g., ethylene or methyl mercaptan, was added to define the role played by fuel impurities. Conclusions are reached as to degradation mechanisms and effects, methods for the elimination of these mechanisms, selection of copper alloy combustion chamber liners, and hydrocarbon fuel purchase specifications.

Completed Beltrami-Michell Formulation in Polar Coordinates

NASA Technical Reports Server (NTRS)

Patnaik, Surya N.; Hopkins, Dale A.

2005-01-01

A set of conditions had not been formulated on the boundary of an elastic continuum since the time of Saint-Venant. This limitation prevented the formulation of a direct stress calculation method in elasticity for a continuum with a displacement boundary condition. The missed condition, referred to as the boundary compatibility condition, is now formulated in polar coordinates. The augmentation of the new condition completes the Beltrami-Michell formulation in polar coordinates. The completed formulation that includes equilibrium equations and a compatibility condition in the field as well as the traction and boundary compatibility condition is derived from the stationary condition of the variational functional of the integrated force method. The new method is illustrated by solving an example of a mixed boundary value problem for mechanical as well as thermal loads.

New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae.

PubMed

Malcova, Ivana; Farkasovsky, Marian; Senohrabkova, Lenka; Vasicova, Pavla; Hasek, Jiri

2016-05-01

Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

System and method for detecting cells or components thereof

DOEpatents

Porter, Marc D [Ames, IA; Lipert, Robert J [Ames, IA; Doyle, Robert T [Ames, IA; Grubisha, Desiree S [Corona, CA; Rahman, Salma [Ames, IA

2009-01-06

A system and method for detecting a detectably labeled cell or component thereof in a sample comprising one or more cells or components thereof, at least one cell or component thereof of which is detectably labeled with at least two detectable labels. In one embodiment, the method comprises: (i) introducing the sample into one or more flow cells of a flow cytometer, (ii) irradiating the sample with one or more light sources that are absorbed by the at least two detectable labels, the absorption of which is to be detected, and (iii) detecting simultaneously the absorption of light by the at least two detectable labels on the detectably labeled cell or component thereof with an array of photomultiplier tubes, which are operably linked to two or more filters that selectively transmit detectable emissions from the at least two detectable labels.

Step-gate polysilicon nanowires field effect transistor compatible with CMOS technology for label-free DNA biosensor.

PubMed

Wenga, G; Jacques, E; Salaün, A-C; Rogel, R; Pichon, L; Geneste, F

2013-02-15

Currently, detection of DNA hybridization using fluorescence-based detection technique requires expensive optical systems and complex bioinformatics tools. Hence, the development of new low cost devices that enable direct and highly sensitive detection stimulates a lot of research efforts. Particularly, devices based on silicon nanowires are emerging as ultrasensitive electrical sensors for the direct detection of biological species thanks to their high surface to volume ratio. In this study, we propose innovative devices using step-gate polycrystalline silicon nanowire FET (poly-Si NW FETs), achieved with simple and low cost fabrication process, and used as ultrasensitive electronic sensor for DNA hybridization. The poly-SiNWs are synthesized using the sidewall spacer formation technique. The detailed fabrication procedure for a step-gate NWFET sensor is described in this paper. No-complementary and complementary DNA sequences were clearly discriminated and detection limit to 1 fM range is observed. This first result using this nano-device is promising for the development of low cost and ultrasensitive polysilicon nanowires based DNA sensors compatible with the CMOS technology. Copyright © 2012 Elsevier B.V. All rights reserved.

Mass spectrometry compatible surfactant for optimized in-gel protein digestion.

PubMed

Saveliev, Sergei V; Woodroofe, Carolyn C; Sabat, Grzegorz; Adams, Christopher M; Klaubert, Dieter; Wood, Keith; Urh, Marjeta

2013-01-15

Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5-2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20-30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment.

Modification of polymers by polymeric additives

NASA Astrophysics Data System (ADS)

Nesterov, A. E.; Lebedev, E. V.

1989-08-01

The conditions for the thermodynamic compatibility of polymers and methods for its enhancement are examined. The study of the influence of various factors on the concentration-temperature limits of compatibility, dispersion stabilisation processes, and methods for the improvement of adhesion between phases in mixtures of thermodynamically incompatible polymers is described. Questions concerning the improvement of the physicomechanical characteristics of polymer dispersions are considered. The bibliography includes 200 references.

Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

PubMed Central

D. M., Jayaseema; Lai, Jiann-Shiun; Hueng, Dueng-Yuan; Chang, Chen

2013-01-01

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs. PMID:23468856

EMC analysis of MOS-1

NASA Astrophysics Data System (ADS)

Ishizawa, Y.; Abe, K.; Shirako, G.; Takai, T.; Kato, H.

The electromagnetic compatibility (EMC) control method, system EMC analysis method, and system test method which have been applied to test the components of the MOS-1 satellite are described. The merits and demerits of the problem solving, specification, and system approaches to EMC control are summarized, and the data requirements of the SEMCAP (specification and electromagnetic compatibility analysis program) computer program for verifying the EMI safety margin of the components are sumamrized. Examples of EMC design are mentioned, and the EMC design process and selection method for EMC critical points are shown along with sample EMC test results.

ALE: automated label extraction from GEO metadata.

PubMed

Giles, Cory B; Brown, Chase A; Ripperger, Michael; Dennis, Zane; Roopnarinesingh, Xiavan; Porter, Hunter; Perz, Aleksandra; Wren, Jonathan D

2017-12-28

NCBI's Gene Expression Omnibus (GEO) is a rich community resource containing millions of gene expression experiments from human, mouse, rat, and other model organisms. However, information about each experiment (metadata) is in the format of an open-ended, non-standardized textual description provided by the depositor. Thus, classification of experiments for meta-analysis by factors such as gender, age of the sample donor, and tissue of origin is not feasible without assigning labels to the experiments. Automated approaches are preferable for this, primarily because of the size and volume of the data to be processed, but also because it ensures standardization and consistency. While some of these labels can be extracted directly from the textual metadata, many of the data available do not contain explicit text informing the researcher about the age and gender of the subjects with the study. To bridge this gap, machine-learning methods can be trained to use the gene expression patterns associated with the text-derived labels to refine label-prediction confidence. Our analysis shows only 26% of metadata text contains information about gender and 21% about age. In order to ameliorate the lack of available labels for these data sets, we first extract labels from the textual metadata for each GEO RNA dataset and evaluate the performance against a gold standard of manually curated labels. We then use machine-learning methods to predict labels, based upon gene expression of the samples and compare this to the text-based method. Here we present an automated method to extract labels for age, gender, and tissue from textual metadata and GEO data using both a heuristic approach as well as machine learning. We show the two methods together improve accuracy of label assignment to GEO samples.

School Foodservice Personnel's Struggle with Using Labels to Identify Whole-Grain Foods

ERIC Educational Resources Information Center

Chu, Yen Li; Orsted, Mary; Marquart, Len; Reicks, Marla

2012-01-01

Objective: To describe how school foodservice personnel use current labeling methods to identify whole-grain products and the influence on purchasing for school meals. Methods: Focus groups explored labeling methods to identify whole-grain products and barriers to incorporating whole-grain foods in school meals. Qualitative analysis procedures and…

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

PubMed Central

Sancey, Lucie; Motto-Ros, Vincent; Kotb, Shady; Wang, Xiaochun; Lux, François; Panczer, Gérard; Yu, Jin; Tillement, Olivier

2014-01-01

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1 to 24 hr after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 μm. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response: elemental, molecular, or cellular. PMID:24962015

Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

PubMed

Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

2009-12-14

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

PubMed

Limsirichaikul, Siripan; Niimi, Atsuko; Fawcett, Heather; Lehmann, Alan; Yamashita, Shunichi; Ogi, Tomoo

2009-03-01

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

Exploiting Sub-threshold and above-threshold characteristics in a silver-enhanced gold nanoparticle based biochip.

PubMed

Liu, Yang; Alocilja, Evangelyn; Chakrabartty, Shantanu

2009-01-01

Silver-enhanced labeling is a technique used in immunochromatographic assays for improving the sensitivity of pathogen detection. In this paper, we employ the silver enhancement approach for constructing a biomolecular transistor that uses a high-density interdigitated electrode to detect rabbit IgG. We show that the response of the biomolecular transistor comprises of: (a) a sub-threshold region where the conductance change is an exponential function of the enhancement time and; (b) an above-threshold region where the conductance change is a linear function with respect to the enhancement time. By exploiting both these regions of operation, it is shown that the silver enhancing time is a reliable indicator of the IgG concentration. The method provides a relatively straightforward alternative to biomolecular signal amplification techniques. The measured results using a biochip prototype fabricated in silicon show that 240 pg/mL rabbit IgG can be detected at the silver enhancing time of 42 min. Also, the biomolecular transistor is compatible with silicon based processing making it ideal for designing integrated CMOS biosensors.

Fidelity of the Integrated Force Method Solution

NASA Technical Reports Server (NTRS)

Hopkins, Dale; Halford, Gary; Coroneos, Rula; Patnaik, Surya

2002-01-01

The theory of strain compatibility of the solid mechanics discipline was incomplete since St. Venant's 'strain formulation' in 1876. We have addressed the compatibility condition both in the continuum and the discrete system. This has lead to the formulation of the Integrated Force Method. A dual Integrated Force Method with displacement as the primal variable has also been formulated. A modest finite element code (IFM/Analyzers) based on the IFM theory has been developed. For a set of standard test problems the IFM results were compared with the stiffness method solutions and the MSC/Nastran code. For the problems IFM outperformed the existing methods. Superior IFM performance is attributed to simultaneous compliance of equilibrium equation and compatibility condition. MSC/Nastran organization expressed reluctance to accept the high fidelity IFM solutions. This report discusses the solutions to the examples. No inaccuracy was detected in the IFM solutions. A stiffness method code with a small programming effort can be improved to reap the many IFM benefits when implemented with the IFMD elements. Dr. Halford conducted a peer-review on the Integrated Force Method. Reviewers' response is included.

Methods of visualizing graphs

DOEpatents

Wong, Pak C.; Mackey, Patrick S.; Perrine, Kenneth A.; Foote, Harlan P.; Thomas, James J.

2008-12-23

Methods for visualizing a graph by automatically drawing elements of the graph as labels are disclosed. In one embodiment, the method comprises receiving node information and edge information from an input device and/or communication interface, constructing a graph layout based at least in part on that information, wherein the edges are automatically drawn as labels, and displaying the graph on a display device according to the graph layout. In some embodiments, the nodes are automatically drawn as labels instead of, or in addition to, the label-edges.

Material Compatibility for Historic Items Decontaminated with ...

EPA Pesticide Factsheets

Report This project continued research of the effects of decontamination methods for biological agents on materials identified as representative of types of irreplaceable objects or works of art found in museums and/or archive settings. In the previous research, surrogate materials were checked for compatibility with four decontamination methods: chlorine dioxide, hydrogen peroxide vapor, methyl bromide, and ethylene oxide gas. This project investigated the effects of gamma irradiation, which has also been shown to be an effective decontamination method for biological agents, on the surrogate test materials.

Automatic labeling of MR brain images through extensible learning and atlas forests.

PubMed

Xu, Lijun; Liu, Hong; Song, Enmin; Yan, Meng; Jin, Renchao; Hung, Chih-Cheng

2017-12-01

Multiatlas-based method is extensively used in MR brain images segmentation because of its simplicity and robustness. This method provides excellent accuracy although it is time consuming and limited in terms of obtaining information about new atlases. In this study, an automatic labeling of MR brain images through extensible learning and atlas forest is presented to address these limitations. We propose an extensible learning model which allows the multiatlas-based framework capable of managing the datasets with numerous atlases or dynamic atlas datasets and simultaneously ensure the accuracy of automatic labeling. Two new strategies are used to reduce the time and space complexity and improve the efficiency of the automatic labeling of brain MR images. First, atlases are encoded to atlas forests through random forest technology to reduce the time consumed for cross-registration between atlases and target image, and a scatter spatial vector is designed to eliminate errors caused by inaccurate registration. Second, an atlas selection method based on the extensible learning model is used to select atlases for target image without traversing the entire dataset and then obtain the accurate labeling. The labeling results of the proposed method were evaluated in three public datasets, namely, IBSR, LONI LPBA40, and ADNI. With the proposed method, the dice coefficient metric values on the three datasets were 84.17 ± 4.61%, 83.25 ± 4.29%, and 81.88 ± 4.53% which were 5% higher than those of the conventional method, respectively. The efficiency of the extensible learning model was evaluated by state-of-the-art methods for labeling of MR brain images. Experimental results showed that the proposed method could achieve accurate labeling for MR brain images without traversing the entire datasets. In the proposed multiatlas-based method, extensible learning and atlas forests were applied to control the automatic labeling of brain anatomies on large atlas datasets or dynamic atlas datasets and obtain accurate results. © 2017 American Association of Physicists in Medicine.

Applications of algebraic topology to compatible spatial discretizations.

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

Bochev, Pavel Blagoveston; Hyman, James M.

We provide a common framework for compatible discretizations using algebraic topology to guide our analysis. The main concept is the natural inner product on cochains, which induces a combinatorial Hodge theory. The framework comprises of mutually consistent operations of differentiation and integration, has a discrete Stokes theorem, and preserves the invariants of the DeRham cohomology groups. The latter allows for an elementary calculation of the kernel of the discrete Laplacian. Our framework provides an abstraction that includes examples of compatible finite element, finite volume and finite difference methods. We describe how these methods result from the choice of a reconstructionmore » operator and when they are equivalent.« less

Uniqueness of the joint measurement and the structure of the set of compatible quantum measurements

NASA Astrophysics Data System (ADS)

Guerini, Leonardo; Terra Cunha, Marcelo

2018-04-01

We address the problem of characterising the compatible tuples of measurements that admit a unique joint measurement. We derive a uniqueness criterion based on the method of perturbations and apply it to show that extremal points of the set of compatible tuples admit a unique joint measurement, while all tuples that admit a unique joint measurement lie in the boundary of such a set. We also provide counter-examples showing that none of these properties are both necessary and sufficient, thus completely describing the relation between the joint measurement uniqueness and the structure of the compatible set. As a by-product of our investigations, we completely characterise the extremal and boundary points of the set of general tuples of measurements and of the subset of compatible tuples.

Detergent-compatible proteases: microbial production, properties, and stain removal analysis.

PubMed

Niyonzima, Francois Niyongabo; More, Sunil

2015-01-01

Proteases are one of the most important commercial enzymes used in various industrial domains such as detergent and leather industries. The alkaline proteases as well as other detergent-compatible enzymes such as lipases and amylases serve now as the key components in detergent formulations. They break down various stains during fabric washing. The search for detergent-compatible proteases with better properties is a continuous exercise. The current trend is to use detergent-compatible proteases that are stable over a wide temperature range. Although the proteases showing stability at elevated pH have the capacity to be used in detergent formulations, their usage can be significant if they are also stable and compatible with detergent and detergent ingredients, and also able to remove protein stains. Despite the existence of some reviews on alkaline proteases, there is no specification for the use of alkaline proteases as detergent additives. The present review describes the detergent-compatible proteases tested as detergent additives. An overview was provided for screening, optimization, purification, and properties of detergent compatible proteases, with an emphasis on the stability and compatibility of the alkaline proteases with the detergent and detergent compounds, as well as stain removal examination methods.

A multi-scale residual-based anti-hourglass control for compatible staggered Lagrangian hydrodynamics

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

Kucharik, M.; Scovazzi, Guglielmo; Shashkov, Mikhail Jurievich

Hourglassing is a well-known pathological numerical artifact affecting the robustness and accuracy of Lagrangian methods. There exist a large number of hourglass control/suppression strategies. In the community of the staggered compatible Lagrangian methods, the approach of sub-zonal pressure forces is among the most widely used. However, this approach is known to add numerical strength to the solution, which can cause potential problems in certain types of simulations, for instance in simulations of various instabilities. To avoid this complication, we have adapted the multi-scale residual-based stabilization typically used in the finite element approach for staggered compatible framework. In this study, wemore » describe two discretizations of the new approach and demonstrate their properties and compare with the method of sub-zonal pressure forces on selected numerical problems.« less

A multi-scale residual-based anti-hourglass control for compatible staggered Lagrangian hydrodynamics

DOE PAGES

Kucharik, M.; Scovazzi, Guglielmo; Shashkov, Mikhail Jurievich; ...

2017-10-28

Hourglassing is a well-known pathological numerical artifact affecting the robustness and accuracy of Lagrangian methods. There exist a large number of hourglass control/suppression strategies. In the community of the staggered compatible Lagrangian methods, the approach of sub-zonal pressure forces is among the most widely used. However, this approach is known to add numerical strength to the solution, which can cause potential problems in certain types of simulations, for instance in simulations of various instabilities. To avoid this complication, we have adapted the multi-scale residual-based stabilization typically used in the finite element approach for staggered compatible framework. In this study, wemore » describe two discretizations of the new approach and demonstrate their properties and compare with the method of sub-zonal pressure forces on selected numerical problems.« less

A cytological approach to study meiotic recombination and chromosome dynamics of Arabidopsis thaliana male meiocytes in three dimensions.

PubMed

Hurel, Aurélie; Phillips, Dylan; Vrielynck, Nathalie; Mézard, Christine; Grelon, Mathilde; Christophorou, Nicolas

2018-04-22

During meiotic prophase I chromosomes undergo dramatic conformational changes that accompany chromosome condensation, pairing and recombination between homologs. These changes include the anchoring of telomeres to the nuclear envelope and their clustering to form a bouquet. In plants, these events have been studied and illustrated in intact meiocytes of large genome species. Arabidopsis thaliana is an excellent genetic model where major molecular pathways that control synapsis and recombination between homologs have been uncovered. Yet the study of chromosome dynamics is hampered by current cytological methods that disrupt the 3D architecture of the nucleus. Here we set up a protocol to preserve the 3D configuration of A. thaliana meiocytes. We showed that this technique is compatible with the use of a variety of antibodies that label structural and recombination proteins and were able to highlight the presence of clustered synapsis initiation centers at the nuclear periphery. By using fluorescence in situ hybridization (FISH) we also studied chromosome behavior during premeiotic G2 and prophase I, revealing the existence of a telomere bouquet during A. thaliana male meiosis. In addition we showed that the number of telomeres in a bouquet and its volume vary greatly thus revealing the complexity of telomere behavior during meiotic prophase I. Finally, by using probes that label subtelomeric regions of individual chromosomes we revealed differential localization behaviors of chromosome ends. Our protocol opens new areas of research to investigate chromosome dynamics in A. thaliana meiocytes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone

PubMed Central

Wenke, Jamie L.; McDonald, W. Hayes; Schey, Kevin L.

2016-01-01

Purpose To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics. Methods Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards. Results Results revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ. Conclusions Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ. PMID:27537260

High-throughput monitoring of major cell functions by means of lensfree video microscopy

PubMed Central

Kesavan, S. Vinjimore; Momey, F.; Cioni, O.; David-Watine, B.; Dubrulle, N.; Shorte, S.; Sulpice, E.; Freida, D.; Chalmond, B.; Dinten, J. M.; Gidrol, X.; Allier, C.

2014-01-01

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 – 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells. PMID:25096726

Java Image I/O for VICAR, PDS, and ISIS

NASA Technical Reports Server (NTRS)

Deen, Robert G.; Levoe, Steven R.

2011-01-01

This library, written in Java, supports input and output of images and metadata (labels) in the VICAR, PDS image, and ISIS-2 and ISIS-3 file formats. Three levels of access exist. The first level comprises the low-level, direct access to the file. This allows an application to read and write specific image tiles, lines, or pixels and to manipulate the label data directly. This layer is analogous to the C-language "VICAR Run-Time Library" (RTL), which is the image I/O library for the (C/C++/Fortran) VICAR image processing system from JPL MIPL (Multimission Image Processing Lab). This low-level library can also be used to read and write labeled, uncompressed images stored in formats similar to VICAR, such as ISIS-2 and -3, and a subset of PDS (image format). The second level of access involves two codecs based on Java Advanced Imaging (JAI) to provide access to VICAR and PDS images in a file-format-independent manner. JAI is supplied by Sun Microsystems as an extension to desktop Java, and has a number of codecs for formats such as GIF, TIFF, JPEG, etc. Although Sun has deprecated the codec mechanism (replaced by IIO), it is still used in many places. The VICAR and PDS codecs allow any program written using the JAI codec spec to use VICAR or PDS images automatically, with no specific knowledge of the VICAR or PDS formats. Support for metadata (labels) is included, but is format-dependent. The PDS codec, when processing PDS images with an embedded VIAR label ("dual-labeled images," such as used for MER), presents the VICAR label in a new way that is compatible with the VICAR codec. The third level of access involves VICAR, PDS, and ISIS Image I/O plugins. The Java core includes an "Image I/O" (IIO) package that is similar in concept to the JAI codec, but is newer and more capable. Applications written to the IIO specification can use any image format for which a plug-in exists, with no specific knowledge of the format itself.

A diagram retrieval method with multi-label learning

NASA Astrophysics Data System (ADS)

Fu, Songping; Lu, Xiaoqing; Liu, Lu; Qu, Jingwei; Tang, Zhi

2015-01-01

In recent years, the retrieval of plane geometry figures (PGFs) has attracted increasing attention in the fields of mathematics education and computer science. However, the high cost of matching complex PGF features leads to the low efficiency of most retrieval systems. This paper proposes an indirect classification method based on multi-label learning, which improves retrieval efficiency by reducing the scope of compare operation from the whole database to small candidate groups. Label correlations among PGFs are taken into account for the multi-label classification task. The primitive feature selection for multi-label learning and the feature description of visual geometric elements are conducted individually to match similar PGFs. The experiment results show the competitive performance of the proposed method compared with existing PGF retrieval methods in terms of both time consumption and retrieval quality.

Evaluation of three methods of platelet labelling.

PubMed

Mortelmans, L; Verbruggen, A; De Roo, M; Vermylen, J

1986-07-01

The study of the kinetics of labelled platelets makes sense only when the platelets preserve their viability after separation and labelling. The separation and labelling procedures described in the manual of two producers of 111In-oxinate (Amersham, Mallinckrodt) have been evaluated by in vitro aggregation tests. The method of Mallinckrodt diminished the aggregation capacities of the thrombocytes. The labelled platelets with normal in vitro aggregation response (Amersham) were tested in vivo in 11 patients who underwent peripheral bypass surgery. The platelet half-life and the platelet accumulation on bypass grafts were checked one week post-operatively. Because of the poor in vivo response of both methods (exponential half-life curve and bad graft visualization), a third method was optimized in our laboratory with good in vitro and in vivo results in 12 patients.

Assessing compatibility of direct detection data: halo-independent global likelihood analyses

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

Gelmini, Graciela B.; Huh, Ji-Haeng; Witte, Samuel J.

2016-10-18

We present two different halo-independent methods to assess the compatibility of several direct dark matter detection data sets for a given dark matter model using a global likelihood consisting of at least one extended likelihood and an arbitrary number of Gaussian or Poisson likelihoods. In the first method we find the global best fit halo function (we prove that it is a unique piecewise constant function with a number of down steps smaller than or equal to a maximum number that we compute) and construct a two-sided pointwise confidence band at any desired confidence level, which can then be comparedmore » with those derived from the extended likelihood alone to assess the joint compatibility of the data. In the second method we define a “constrained parameter goodness-of-fit” test statistic, whose p-value we then use to define a “plausibility region” (e.g. where p≥10%). For any halo function not entirely contained within the plausibility region, the level of compatibility of the data is very low (e.g. p<10%). We illustrate these methods by applying them to CDMS-II-Si and SuperCDMS data, assuming dark matter particles with elastic spin-independent isospin-conserving interactions or exothermic spin-independent isospin-violating interactions.« less

Fluorogenic Behaviour of the Hetero-Diels-Alder Ligation of 5-Alkoxyoxazoles with Maleimides and their Applications.

PubMed

Renault, Kévin; Jouanno, Laurie-Anne; Lizzul-Jurse, Antoine; Renard, Pierre-Yves; Sabot, Cyrille

2016-12-19

Fluorogenic reactions are largely underrepresented in the toolbox of chemoselective ligations despite their tremendous potential, particularly in chemical biology and biochemistry. In this respect, we have investigated in full detail the fluorescence behaviour of the azaphthalamide, a scaffold which is generated through a hetero-Diels-Alder reaction of 5-alkoxyoxazole and maleimide derivatives under mild conditions that are compatible with, among others, peptide chemistry. The scope and limitations of such a fluorogenic labelling strategy were examined through four distinct applications, which target enzymatic activities or bioorthogonal reactions. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Accretion of biopsy specimens of vaginal adenosis from patients exposed in utero to diethylstilbestrol, when transplanted to athymic nude mice.

PubMed

Pienkowski, M M; Mann, L C; Rosloniec, E F; Welsch, C W

1979-03-01

Vaginal adenosis biopsy specimens from 10 patients exposed in utero to diethylstilbestrol were transplanted for 30 days into athymic (nude) mice. Almost all grafts were recovered, and they had morphologic features closely resembling those of the original biopsy specimens, i.e., cystic, complex, and simple occult glands covered mainly with an endocervical type of epithelium showing extensive squamous metaplasia. Autoradiographic analysis of these grafts after pulse administration of [3H]thymidine into the mice revealed extensive labeling of epithelial cells. These results imply that female athymic (nude) mice are compatible hosts for accretion of the human adenosis.

An underlap field-effect transistor for electrical detection of influenza

NASA Astrophysics Data System (ADS)

Lee, Kwang-Won; Choi, Sung-Jin; Ahn, Jae-Hyuk; Moon, Dong-Il; Park, Tae Jung; Lee, Sang Yup; Choi, Yang-Kyu

2010-01-01

An underlap channel-embedded field-effect transistor (FET) is proposed for label-free biomolecule detection. Specifically, silica binding protein fused with avian influenza (AI) surface antigen and avian influenza antibody (anti-AI) were designed as a receptor molecule and a target material, respectively. The drain current was significantly decreased after the binding of negatively charged anti-AI on the underlap channel. A set of control experiments supports that only the biomolecules on the underlap channel effectively modulate the drain current. With the merits of a simple fabrication process, complementary metal-oxide-semiconductor compatibility, and enhanced sensitivity, the underlap FET could be a promising candidate for a chip-based biosensor.

A Method for Identifying Small-Molecule Aggregators Using Photonic Crystal Biosensor Microplates

PubMed Central

Chan, Leo L.; Lidstone, Erich A.; Finch, Kristin E.; Heeres, James T.; Hergenrother, Paul J.; Cunningham, Brian T.

2010-01-01

Small molecules identified through high-throughput screens are an essential element in pharmaceutical discovery programs. It is now recognized that a substantial fraction of small molecules exhibit aggregating behavior leading to false positive results in many screening assays, typically due to nonspecific attachment to target proteins. Therefore, the ability to efficiently identify compounds within a screening library that aggregate can streamline the screening process by eliminating unsuitable molecules from further consideration. In this work, we show that photonic crystal (PC) optical biosensor microplate technology can be used to identify and quantify small-molecule aggregation. A group of aggregators and nonaggregators were tested using the PC technology, and measurements were compared with those gathered by three alternative methods: dynamic light scattering (DLS), an α-chymotrypsin colorimetric assay, and scanning electron microscopy (SEM). The PC biosensor measurements of aggregation were confirmed by visual observation using SEM, and were in general agreement with the α-chymotrypsin assay. DLS measurements, in contrast, demonstrated inconsistent readings for many compounds that are found to form aggregates in shapes, very different from the classical spherical particles assumed in DLS modeling. As a label-free detection method, the PC biosensor aggregation assay is simple to implement and provides a quantitative direct measurement of the mass density of material adsorbed to the transducer surface, whereas the microplate-based sensor format enables compatibility with high-throughput automated liquid-handling methods used in pharmaceutical screening. PMID:20930952

Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results

PubMed Central

Bordag, Natalie; Janakiraman, Vijay; Nachtigall, Jonny; González Maldonado, Sandra; Bethan, Bianca; Laine, Jean-Philippe; Fux, Elie

2016-01-01

The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data. PMID:27438065

Hierarchical Multi-atlas Label Fusion with Multi-scale Feature Representation and Label-specific Patch Partition

PubMed Central

Wu, Guorong; Kim, Minjeong; Sanroma, Gerard; Wang, Qian; Munsell, Brent C.; Shen, Dinggang

2014-01-01

Multi-atlas patch-based label fusion methods have been successfully used to improve segmentation accuracy in many important medical image analysis applications. In general, to achieve label fusion a single target image is first registered to several atlas images, after registration a label is assigned to each target point in the target image by determining the similarity between the underlying target image patch (centered at the target point) and the aligned image patch in each atlas image. To achieve the highest level of accuracy during the label fusion process it’s critical the chosen patch similarity measurement accurately captures the tissue/shape appearance of the anatomical structure. One major limitation of existing state-of-the-art label fusion methods is that they often apply a fixed size image patch throughout the entire label fusion procedure. Doing so may severely affect the fidelity of the patch similarity measurement, which in turn may not adequately capture complex tissue appearance patterns expressed by the anatomical structure. To address this limitation, we advance state-of-the-art by adding three new label fusion contributions: First, each image patch now characterized by a multi-scale feature representation that encodes both local and semi-local image information. Doing so will increase the accuracy of the patch-based similarity measurement. Second, to limit the possibility of the patch-based similarity measurement being wrongly guided by the presence of multiple anatomical structures in the same image patch, each atlas image patch is further partitioned into a set of label-specific partial image patches according to the existing labels. Since image information has now been semantically divided into different patterns, these new label-specific atlas patches make the label fusion process more specific and flexible. Lastly, in order to correct target points that are mislabeled during label fusion, a hierarchically approach is used to improve the label fusion results. In particular, a coarse-to-fine iterative label fusion approach is used that gradually reduces the patch size. To evaluate the accuracy of our label fusion approach, the proposed method was used to segment the hippocampus in the ADNI dataset and 7.0 tesla MR images, sub-cortical regions in LONI LBPA40 dataset, mid-brain regions in SATA dataset from MICCAI 2013 segmentation challenge, and a set of key internal gray matter structures in IXI dataset. In all experiments, the segmentation results of the proposed hierarchical label fusion method with multi-scale feature representations and label-specific atlas patches are more accurate than several well-known state-of-the-art label fusion methods. PMID:25463474

Multi-Atlas Based Segmentation of Brainstem Nuclei from MR Images by Deep Hyper-Graph Learning.

PubMed

Dong, Pei; Guo, Yangrong; Gao, Yue; Liang, Peipeng; Shi, Yonghong; Wang, Qian; Shen, Dinggang; Wu, Guorong

2016-10-01

Accurate segmentation of brainstem nuclei (red nucleus and substantia nigra) is very important in various neuroimaging applications such as deep brain stimulation and the investigation of imaging biomarkers for Parkinson's disease (PD). Due to iron deposition during aging, image contrast in the brainstem is very low in Magnetic Resonance (MR) images. Hence, the ambiguity of patch-wise similarity makes the recently successful multi-atlas patch-based label fusion methods have difficulty to perform as competitive as segmenting cortical and sub-cortical regions from MR images. To address this challenge, we propose a novel multi-atlas brainstem nuclei segmentation method using deep hyper-graph learning. Specifically, we achieve this goal in three-fold. First , we employ hyper-graph to combine the advantage of maintaining spatial coherence from graph-based segmentation approaches and the benefit of harnessing population priors from multi-atlas based framework. Second , besides using low-level image appearance, we also extract high-level context features to measure the complex patch-wise relationship. Since the context features are calculated on a tentatively estimated label probability map, we eventually turn our hyper-graph learning based label propagation into a deep and self-refining model. Third , since anatomical labels on some voxels (usually located in uniform regions) can be identified much more reliably than other voxels (usually located at the boundary between two regions), we allow these reliable voxels to propagate their labels to the nearby difficult-to-label voxels. Such hierarchical strategy makes our proposed label fusion method deep and dynamic. We evaluate our proposed label fusion method in segmenting substantia nigra (SN) and red nucleus (RN) from 3.0 T MR images, where our proposed method achieves significant improvement over the state-of-the-art label fusion methods.

Al18F-Labeling Of Heat-Sensitive Biomolecules for Positron Emission Tomography Imaging.

PubMed

Cleeren, Frederik; Lecina, Joan; Ahamed, Muneer; Raes, Geert; Devoogdt, Nick; Caveliers, Vicky; McQuade, Paul; Rubins, Daniel J; Li, Wenping; Verbruggen, Alfons; Xavier, Catarina; Bormans, Guy

2017-01-01

Positron emission tomography (PET) using radiolabeled biomolecules is a translational molecular imaging technology that is increasingly used in support of drug development. Current methods for radiolabeling biomolecules with fluorine-18 are laborious and require multistep procedures with moderate labeling yields. The Al 18 F-labeling strategy involves chelation in aqueous medium of aluminum mono[ 18 F]fluoride ({Al 18 F} 2+ ) by a suitable chelator conjugated to a biomolecule. However, the need for elevated temperatures (100-120 °C) required for the chelation reaction limits its widespread use. Therefore, we designed a new restrained complexing agent (RESCA) for application of the AlF strategy at room temperature. Methods. The new chelator RESCA was conjugated to three relevant biologicals and the constructs were labeled with {Al 18 F} 2+ to evaluate the generic applicability of the one-step Al 18 F-RESCA-method. Results. We successfully labeled human serum albumin with excellent radiochemical yields in less than 30 minutes and confirmed in vivo stability of the Al 18 F-labeled protein in rats. In addition, we efficiently labeled nanobodies targeting the Kupffer cell marker CRIg, and performed µPET studies in healthy and CRIg deficient mice to demonstrate that the proposed radiolabeling method does not affect the functional integrity of the protein. Finally, an affibody targeting HER2 (PEP04314) was labeled site-specifically, and the distribution profile of (±)-[ 18 F]AlF(RESCA)-PEP04314 in a rhesus monkey was compared with that of [ 18 F]AlF(NOTA)-PEP04314 using whole-body PET/CT. Conclusion. This generic radiolabeling method has the potential to be a kit-based fluorine-18 labeling strategy, and could have a large impact on PET radiochemical space, potentially enabling the development of many new fluorine-18 labeled protein-based radiotracers.

Multi-label literature classification based on the Gene Ontology graph.

PubMed

Jin, Bo; Muller, Brian; Zhai, Chengxiang; Lu, Xinghua

2008-12-08

The Gene Ontology is a controlled vocabulary for representing knowledge related to genes and proteins in a computable form. The current effort of manually annotating proteins with the Gene Ontology is outpaced by the rate of accumulation of biomedical knowledge in literature, which urges the development of text mining approaches to facilitate the process by automatically extracting the Gene Ontology annotation from literature. The task is usually cast as a text classification problem, and contemporary methods are confronted with unbalanced training data and the difficulties associated with multi-label classification. In this research, we investigated the methods of enhancing automatic multi-label classification of biomedical literature by utilizing the structure of the Gene Ontology graph. We have studied three graph-based multi-label classification algorithms, including a novel stochastic algorithm and two top-down hierarchical classification methods for multi-label literature classification. We systematically evaluated and compared these graph-based classification algorithms to a conventional flat multi-label algorithm. The results indicate that, through utilizing the information from the structure of the Gene Ontology graph, the graph-based multi-label classification methods can significantly improve predictions of the Gene Ontology terms implied by the analyzed text. Furthermore, the graph-based multi-label classifiers are capable of suggesting Gene Ontology annotations (to curators) that are closely related to the true annotations even if they fail to predict the true ones directly. A software package implementing the studied algorithms is available for the research community. Through utilizing the information from the structure of the Gene Ontology graph, the graph-based multi-label classification methods have better potential than the conventional flat multi-label classification approach to facilitate protein annotation based on the literature.

Multiple tag labeling method for DNA sequencing

DOEpatents

Mathies, Richard A.; Huang, Xiaohua C.; Quesada, Mark A.

1995-01-01

A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

Multi-label learning with fuzzy hypergraph regularization for protein subcellular location prediction.

PubMed

Chen, Jing; Tang, Yuan Yan; Chen, C L Philip; Fang, Bin; Lin, Yuewei; Shang, Zhaowei

2014-12-01

Protein subcellular location prediction aims to predict the location where a protein resides within a cell using computational methods. Considering the main limitations of the existing methods, we propose a hierarchical multi-label learning model FHML for both single-location proteins and multi-location proteins. The latent concepts are extracted through feature space decomposition and label space decomposition under the nonnegative data factorization framework. The extracted latent concepts are used as the codebook to indirectly connect the protein features to their annotations. We construct dual fuzzy hypergraphs to capture the intrinsic high-order relations embedded in not only feature space, but also label space. Finally, the subcellular location annotation information is propagated from the labeled proteins to the unlabeled proteins by performing dual fuzzy hypergraph Laplacian regularization. The experimental results on the six protein benchmark datasets demonstrate the superiority of our proposed method by comparing it with the state-of-the-art methods, and illustrate the benefit of exploiting both feature correlations and label correlations.

Lesson plan profile of senior high school biology teachers in Subang

NASA Astrophysics Data System (ADS)

Rohayati, E.; Diana, S. W.; Priyandoko, D.

2018-05-01

Lesson plan have important role for biology teachers in teaching and learning process. The aim of this study was intended to gain an overview of lesson plan of biology teachers’ at Senior High Schools in Subang which were the members of biology teachers association in Subang. The research method was descriptive method. Data was collected from 30 biology teachers. The result of study showed that lesson plan profile in terms of subject’s identity had good category with 83.33 % of average score. Analysis on basic competence in fair category with 74.45 % of average score. The compatibility of method/strategy was in fair category with average score 72.22 %. The compatibility of instrument, media, and learning resources in fair category with 71.11 % of average score. Learning scenario was in good category with 77.00 % of average score. The compatibility of evaluation was in low category with 56.39 % of average score. It can be concluded that biology teachers in Subang were good enough in making lesson plan, however in terms of the compatibility of evaluation needed to be fixed. Furthermore, teachers’ training for biology teachers’ association was recommended to increasing teachers’ skill to be professional teachers.

Mass spectrometry-compatible silver staining of histones resolved on acetic acid-urea-Triton PAGE.

PubMed

Pramod, Khare Satyajeet; Bharat, Khade; Sanjay, Gupta

2009-05-01

Acetic acid-Urea-Triton (AUT) PAGE is commonly used method to separate histone variants and their post-translationally modified forms. Coomassie staining is the preferred method for protein visualization; however, its sensitivity is less than that of silver staining. Though silver staining of histones in AUT-PAGE has been reported, the method is time-consuming, dependent on prior staining by Amido black and has not been reported suitable for mass spectrometry. Here, we propose 'SDS-Silver' method for rapid, sensitive and mass spectrometry-compatible staining of histones resolved on AUT-PAGE.

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

PubMed Central

Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

2000-01-01

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

[Gastric emptying of a solid-liquid meal in normal subjects: validity of the labeling (99mTc) of chicken liver by a multipuncture technic].

PubMed

Hostein, J; Capony, P; Busquet, G; Bost, R; Fournet, J

1985-04-01

For gastric emptying studies of a solid-liquid meal by the scintigraphic method, a valid isotope labeling method for each phase of the meal must be obtained. The aim of this study was to validate a simple chicken liver labeling method in normal subjects by multipuncture technic with 99mtechnetium. Labeling according to Meyer's method was chosen as a reference. Simultaneously, a study of the quality of liquid phase labeling by 111indium was done. The labeling process quality for each phase of the meal was assessed: a) in vitro, after incubation of the meal with human gastric juice (n = 12); b) in vivo, after meal ingestion and sequential collection of gastric contents by aspiration (n = 4). Furthermore, in 8 healthy volunteers, gastric emptying curves of the solid and liquid phases of the meal were determined scintigraphically and compared. Our results showed: a) for the solid phase: a good specificity of the marker, which was assessed in vitro and in vivo, after liver labeling with multipuncture technique (89 p. 100 and 92 p. 100 after 180 min, respectively); b) for the liquid phase: a good specificity of the marker in vitro and a poor specificity in vivo (82 p. 100 and 27 p. 100 after 180 min, respectively); c) similar half-gastric emptying times and cumulative percentages for the solid and liquid phases with both liver labeling methods. In conclusion, the multipuncture technique for chicken liver labeling may be used for gastric emptying studies in humans.

Weakly supervised image semantic segmentation based on clustering superpixels

NASA Astrophysics Data System (ADS)

Yan, Xiong; Liu, Xiaohua

2018-04-01

In this paper, we propose an image semantic segmentation model which is trained from image-level labeled images. The proposed model starts with superpixel segmenting, and features of the superpixels are extracted by trained CNN. We introduce a superpixel-based graph followed by applying the graph partition method to group correlated superpixels into clusters. For the acquisition of inter-label correlations between the image-level labels in dataset, we not only utilize label co-occurrence statistics but also exploit visual contextual cues simultaneously. At last, we formulate the task of mapping appropriate image-level labels to the detected clusters as a problem of convex minimization. Experimental results on MSRC-21 dataset and LableMe dataset show that the proposed method has a better performance than most of the weakly supervised methods and is even comparable to fully supervised methods.

Advanced surface-enhanced Raman gene probe systems and methods thereof

DOEpatents

Vo-Dinh, Tuan

2001-01-01

The subject invention is a series of methods and systems for using the Surface-Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material comprising the steps of immobilizing SER-labeled hybridized target oligonucleotide material on a support means, wherein the SER-labeled hybridized target oligonucleotide material comprise a SER label attached either to a target oligonucleotide of unknown sequence or to a gene probe of known sequence complementary to the target oligonucleotide sequence, the SER label is unique for the target oligonucleotide strands of a particular sequence wherein the SER-labeled oligonucleotide is hybridized to its complementary oligonucleotide strand, then the support means having the SER-labeled hybridized target oligonucleotide material adsorbed thereon is SERS activated with a SERS activating means, then the support means is analyzed.

49 CFR Appendix B to Part 173 - Procedure for Testing Chemical Compatibility and Rate of Permeation in Plastic Packaging and...

Code of Federal Regulations, 2014 CFR

2014-10-01

... Rate of Permeation in Plastic Packaging and Receptacles B Appendix B to Part 173 Transportation Other..., App. B Appendix B to Part 173—Procedure for Testing Chemical Compatibility and Rate of Permeation in... °C. (64 °F.) b. Test Method 2: 28 days at a temperature no lower than 50 °C. (122 °F.) c. Test Method...

[Mechanical strength and mechano-compatibility of tissue-engineered bones].

PubMed

Tanaka, Shigeo

2016-01-01

Current artificial bones made of metals and ceramics may be replaced around a decade after implantation due to its low durability, which is brought on by a large difference from the host bone in mechanical properties, i.e., low mechano-compatibility. On the other hand, tissue engineering could be a solution with regeneration of bone tissues from stem cells in vitro. However, there are still some problems to realize exactly the same mechanical properties as those of real bone. This paper introduces the technical background of bone tissue engineering and discusses possible methods for installation of mechano-compatibility into a regenerative bone. At the end, future directions toward the realization of ideal mechano-compatible regenerative bone are proposed.

Efficacy-oriented compatibility for component-based Chinese medicine

PubMed Central

Zhang, Jun-hua; Zhu, Yan; Fan, Xiao-hui; Zhang, Bo-li

2015-01-01

Single-target drugs have not achieved satisfactory therapeutic effects for complex diseases involving multiple factors. Instead, innovations in recent drug research and development have revealed the emergence of compound drugs, such as cocktail therapies and “polypills”, as the frontier in new drug development. A traditional Chinese medicine (TCM) prescription that is usually composed of several medicinal herbs can serve a typical representative of compound medicines. Although the traditional compatibility theory of TCM cannot be well expressed using modern scientific language nowadays, the fundamental purpose of TCM compatibility can be understood as promoting efficacy and reducing toxicity. This paper introduces the theory and methods of efficacy-oriented compatibility for developing component-based Chinese medicines. PMID:25864650

Applying Standard Independent Verification and Validation (IVV) Techniques Within an Agile Framework: Is There a Compatibility Issue?

NASA Technical Reports Server (NTRS)

Dabney, James B.; Arthur, James Douglas

2017-01-01

Agile methods have gained wide acceptance over the past several years, to the point that they are now a standard management and execution approach for small-scale software development projects. While conventional Agile methods are not generally applicable to large multi-year and mission-critical systems, Agile hybrids are now being developed (such as SAFe) to exploit the productivity improvements of Agile while retaining the necessary process rigor and coordination needs of these projects. From the perspective of Independent Verification and Validation (IVV), however, the adoption of these hybrid Agile frameworks is becoming somewhat problematic. Hence, we find it prudent to question the compatibility of conventional IVV techniques with (hybrid) Agile practices.This paper documents our investigation of (a) relevant literature, (b) the modification and adoption of Agile frameworks to accommodate the development of large scale, mission critical systems, and (c) the compatibility of standard IVV techniques within hybrid Agile development frameworks. Specific to the latter, we found that the IVV methods employed within a hybrid Agile process can be divided into three groups: (1) early lifecycle IVV techniques that are fully compatible with the hybrid lifecycles, (2) IVV techniques that focus on tracing requirements, test objectives, etc. are somewhat incompatible, but can be tailored with a modest effort, and (3) IVV techniques involving an assessment requiring artifact completeness that are simply not compatible with hybrid Agile processes, e.g., those that assume complete requirement specification early in the development lifecycle.

Structural analysis of N-glycans by the glycan-labeling method using 3-aminoquinoline-based liquid matrix in negative-ion MALDI-MS.

PubMed

Nishikaze, Takashi; Kaneshiro, Kaoru; Kawabata, Shin-ichirou; Tanaka, Koichi

2012-11-06

Negative-ion fragmentation of underivatized N-glycans has been proven to be more informative than positive-ion fragmentation. Fluorescent labeling via reductive amination is often employed for glycan analysis, but little is known about the influence of the labeling group on negative-ion fragmentation. We previously demonstrated that the on-target glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid (3AQ/CHCA) liquid matrix enables highly sensitive, rapid, and quantitative N-glycan profiling analysis. The current study investigates the suitability of 3AQ-labeled N-glycans for structural analysis based on negative-ion collision-induced dissociation (CID) spectra. 3AQ-labeled N-glycans exhibited simple and informative CID spectra similar to those of underivatized N-glycans, with product ions due to cross-ring cleavages of the chitobiose core and ions specific to two antennae (D and E ions). The interpretation of diagnostic fragment ions suggested for underivatized N-glycans could be directly applied to the 3AQ-labeled N-glycans. However, fluorescently labeled N-glycans by conventional reductive amination, such as 2-aminobenzamide (2AB)- and 2-pyrydilamine (2PA)-labeled N-glycans, exhibited complicated CID spectra consisting of numerous signals formed by dehydration and multiple cleavages. The complicated spectra of 2AB- and 2PA-labeled N-glycans was found to be due to their open reducing-terminal N-acetylglucosamine (GlcNAc) ring, rather than structural differences in the labeling group in the N-glycan derivative. Finally, as an example, the on-target 3AQ labeling method followed by negative-ion CID was applied to structurally analyze neutral N-glycans released from human epidermal growth factor receptor type 2 (HER2) protein. The glycan-labeling method using 3AQ-based liquid matrix should facilitate highly sensitive quantitative and qualitative analyses of glycans.

Multi-atlas segmentation with joint label fusion and corrective learning—an open source implementation

PubMed Central

Wang, Hongzhi; Yushkevich, Paul A.

2013-01-01

Label fusion based multi-atlas segmentation has proven to be one of the most competitive techniques for medical image segmentation. This technique transfers segmentations from expert-labeled images, called atlases, to a novel image using deformable image registration. Errors produced by label transfer are further reduced by label fusion that combines the results produced by all atlases into a consensus solution. Among the proposed label fusion strategies, weighted voting with spatially varying weight distributions derived from atlas-target intensity similarity is a simple and highly effective label fusion technique. However, one limitation of most weighted voting methods is that the weights are computed independently for each atlas, without taking into account the fact that different atlases may produce similar label errors. To address this problem, we recently developed the joint label fusion technique and the corrective learning technique, which won the first place of the 2012 MICCAI Multi-Atlas Labeling Challenge and was one of the top performers in 2013 MICCAI Segmentation: Algorithms, Theory and Applications (SATA) challenge. To make our techniques more accessible to the scientific research community, we describe an Insight-Toolkit based open source implementation of our label fusion methods. Our implementation extends our methods to work with multi-modality imaging data and is more suitable for segmentation problems with multiple labels. We demonstrate the usage of our tools through applying them to the 2012 MICCAI Multi-Atlas Labeling Challenge brain image dataset and the 2013 SATA challenge canine leg image dataset. We report the best results on these two datasets so far. PMID:24319427

Multilabel learning via random label selection for protein subcellular multilocations prediction.

PubMed

Wang, Xiao; Li, Guo-Zheng

2013-01-01

Prediction of protein subcellular localization is an important but challenging problem, particularly when proteins may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular localization methods are only used to deal with the single-location proteins. In the past few years, only a few methods have been proposed to tackle proteins with multiple locations. However, they only adopt a simple strategy, that is, transforming the multilocation proteins to multiple proteins with single location, which does not take correlations among different subcellular locations into account. In this paper, a novel method named random label selection (RALS) (multilabel learning via RALS), which extends the simple binary relevance (BR) method, is proposed to learn from multilocation proteins in an effective and efficient way. RALS does not explicitly find the correlations among labels, but rather implicitly attempts to learn the label correlations from data by augmenting original feature space with randomly selected labels as its additional input features. Through the fivefold cross-validation test on a benchmark data set, we demonstrate our proposed method with consideration of label correlations obviously outperforms the baseline BR method without consideration of label correlations, indicating correlations among different subcellular locations really exist and contribute to improvement of prediction performance. Experimental results on two benchmark data sets also show that our proposed methods achieve significantly higher performance than some other state-of-the-art methods in predicting subcellular multilocations of proteins. The prediction web server is available at >http://levis.tongji.edu.cn:8080/bioinfo/MLPred-Euk/ for the public usage.

Multiple tag labeling method for DNA sequencing

DOEpatents

Mathies, R.A.; Huang, X.C.; Quesada, M.A.

1995-07-25

A DNA sequencing method is described which uses single lane or channel electrophoresis. Sequencing fragments are separated in the lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radioisotope labels. 5 figs.

Automatic prevention of label overlap

DOT National Transportation Integrated Search

1976-03-01

The project comprised a number of simulation exercises : designed to evaluate methods of either preventing or : resolving the problems likely to be caused by label overlap on : Labelled Plan Displays (LPD). The automatic prevention of : label overlap...

Photonic-plasmonic hybrid single-molecule nanosensor measures the effect of fluorescent labels on DNA-protein dynamics

PubMed Central

Liang, Feng; Guo, Yuzheng; Hou, Shaocong; Quan, Qimin

2017-01-01

Current methods to study molecular interactions require labeling the subject molecules with fluorescent reporters. However, the effect of the fluorescent reporters on molecular dynamics has not been quantified because of a lack of alternative methods. We develop a hybrid photonic-plasmonic antenna-in-a-nanocavity single-molecule biosensor to study DNA-protein dynamics without using fluorescent labels. Our results indicate that the fluorescein and fluorescent protein labels decrease the interaction between a single DNA and a protein due to weakened electrostatic interaction. Although the study is performed on the DNA-XPA system, the conclusion has a general implication that the traditional fluorescent labeling methods might be misestimating the molecular interactions. PMID:28560341

Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling.

PubMed

Neville, David C A; Coquard, Virginie; Priestman, David A; te Vruchte, Danielle J M; Sillence, Daniel J; Dwek, Raymond A; Platt, Frances M; Butters, Terry D

2004-08-15

Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.

Cell Kinetic and Histomorphometric Analysis of Microgravitational Osteopenia: PARE.03B

NASA Technical Reports Server (NTRS)

Roberts, W. Eugene; Garetto, Lawrence P.

1998-01-01

Previous methods of identifying cells undergoing DNA synthesis (S-phase) utilized 3H-thymidine (3HT) autoradiography. 5-Bromo-2'-deoxyuridine (BrdU) immunohistochemistry is a nonradioactive alternative method. This experiment compared the two methods using the nuclear volume model for osteoblast histogenesis in two different embedding media. Twenty Sprague-Dawley rats were used, with half receiving 3HT (1 micro-Ci/g) and the other half BrdU (50 micro-g/g). Condyles were embedded (one side in paraffin, the other in plastic) and S-phase nuclei were identified using either autoradiography or immunohistochemistry. The fractional distribution of preosteoblast cell types and the percentage of labeled cells (within each cell fraction and label index) were calculated and expressed as mean +/- standard error. Chi-Square analysis showed only a minor difference in the fractional distribution of cell types. However, there were,significant differences (p less than 0.05) by ANOVA, in the nuclear labeling of specific cell types. With the exception of the less-differentiated A+A' cells, more BrdU label was consistently detected in paraffin than in plastic-embedded sections. In general, more nuclei were labeled with 3H-thymidine than with BrdU in both types of embedding media (Fig 2.). Labeling index data (labeled cells/total cells sampled x 100) indicated that BrdU in paraffin, but not plastic gave the same results as 3HT in either embedding method. Thus, we conclude that the two labeling methods do not yield the same results.

Alcohol Warning Label Awareness and Attention: A Multi-method Study.

PubMed

Pham, Cuong; Rundle-Thiele, Sharyn; Parkinson, Joy; Li, Shanshi

2018-01-01

Evaluation of alcohol warning labels requires careful consideration ensuring that research captures more than awareness given that labels may not be prominent enough to attract attention. This study investigates attention of current in market alcohol warning labels and examines whether attention can be enhanced through theoretically informed design. Attention scores obtained through self-report methods are compared to objective measures (eye-tracking). A multi-method experimental design was used delivering four conditions, namely control, colour, size and colour and size. The first study (n = 559) involved a self-report survey to measure attention. The second study (n = 87) utilized eye-tracking to measure fixation count and duration and time to first fixation. Analysis of Variance (ANOVA) was utilized. Eye-tracking identified that 60% of participants looked at the current in market alcohol warning label while 81% looked at the optimized design (larger and red). In line with observed attention self-reported attention increased for the optimized design. The current study casts doubt on dominant practices (largely self-report), which have been used to evaluate alcohol warning labels. Awareness cannot be used to assess warning label effectiveness in isolation in cases where attention does not occur 100% of the time. Mixed methods permit objective data collection methodologies to be triangulated with surveys to assess warning label effectiveness. Attention should be incorporated as a measure in warning label effectiveness evaluations. Colour and size changes to the existing Australian warning labels aided by theoretically informed design increased attention. © The Author 2017. Medical Council on Alcohol and Oxford University Press. All rights reserved.

Synthesis and Labeling of RNA In Vitro

PubMed Central

Huang, Chao; Yu, Yi-Tao

2013-01-01

This unit discusses several methods for generating large amounts of uniformly labeled, end-labeled, and site-specifically labeled RNAs in vitro. The methods involve a number of experimental procedures, including RNA transcription, 5′ dephosphorylation and rephosphorylation, 3′ terminal nucleotide addition (via ligation), site-specific RNase H cleavage directed by 2′-O-methyl RNA-DNA chimeras, and 2-piece splint ligation. The applications of these RNA radiolabeling approaches are also discussed. PMID:23547015

Determination of dye/protein ratios in a labeling reaction between a cyanine dye and bovine serum albumin by micellar electrokinetic chromatography using a diode laser-induced fluorescence detection.

PubMed

Jing, Peng; Kaneta, Takashi; Imasaka, Totaro

2002-08-01

The degree of labeling, i.e., dye/protein ratio (D/P) is important for characterizing properties of dye labeling with proteins. A method for the determination of this ratio between a fluorescent cyanine dye and bovine serum albumin (BSA), based on the separation of the labeling mixture using micellar electrokinetic chromatography with diode laser-induced fluorescence detection, is described. Two methods for the determination of D/P were examined in this study. In these methods, a hydrolysis product and impurities, which are usually unfavorable compounds that are best excluded for protein analysis, were utilized to determine the amounts of dye bound to BSA. One is a direct method in which a ratio of the peak area of BSA to the total peak area of all the products produced in the labeling reaction was used for determining the average number of dye molecules bound to a single BSA molecule. The other is an indirect determination, which is based on diminution of all peak areas related to the products except for the labeled BSA. These methods were directly compared by means of a spectrophotometric method. The experimental results show that the indirect method is both reliable and sensitive. Therefore, D/P values can be determined at trace levels using the indirect method.

Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals

PubMed Central

Nießner, Christine; Denzau, Susanne; Malkemper, Erich Pascal; Gross, Julia Christina; Burda, Hynek; Winklhofer, Michael; Peichl, Leo

2016-01-01

Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown, and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae, and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein. PMID:26898837

Tunable and noncytotoxic PET/SPECT-MRI multimodality imaging probes using colloidally stable ligand-free superparamagnetic iron oxide nanoparticles

PubMed Central

Pham, TH Nguyen; Lengkeek, Nigel A; Greguric, Ivan; Kim, Byung J; Pellegrini, Paul A; Bickley, Stephanie A; Tanudji, Marcel R; Jones, Stephen K; Hawkett, Brian S; Pham, Binh TT

2017-01-01

Physiologically stable multimodality imaging probes for positron emission tomography/single-photon emission computed tomography (PET/SPECT)-magnetic resonance imaging (MRI) were synthesized using the superparamagnetic maghemite iron oxide (γ-Fe2O3) nanoparticles (SPIONs). The SPIONs were sterically stabilized with a finely tuned mixture of diblock copolymers with either methoxypolyethylene glycol (MPEG) or primary amine NH2 end groups. The radioisotope for PET or SPECT imaging was incorporated with the SPIONs at high temperature. 57Co2+ ions with a long half-life of 270.9 days were used as a model for the radiotracer to study the kinetics of radiolabeling, characterization, and the stability of the radiolabeled SPIONs. Radioactive 67Ga3+ and Cu2+-labeled SPIONs were also produced successfully using the optimized conditions from the 57Co2+-labeling process. No free radioisotopes were detected in the aqueous phase for the radiolabeled SPIONs 1 week after dispersion in phosphate-buffered saline (PBS). All labeled SPIONs were not only well dispersed and stable under physiological conditions but also noncytotoxic in vitro. The ability to design and produce physiologically stable radiolabeled magnetic nanoparticles with a finely controlled number of functionalizable end groups on the SPIONs enables the generation of a desirable and biologically compatible multimodality PET/SPECT-MRI agent on a single T2 contrast MRI probe. PMID:28184160

Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals.

PubMed

Nießner, Christine; Denzau, Susanne; Malkemper, Erich Pascal; Gross, Julia Christina; Burda, Hynek; Winklhofer, Michael; Peichl, Leo

2016-02-22

Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown, and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae, and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein.

Couple Graph Based Label Propagation Method for Hyperspectral Remote Sensing Data Classification

NASA Astrophysics Data System (ADS)

Wang, X. P.; Hu, Y.; Chen, J.

2018-04-01

Graph based semi-supervised classification method are widely used for hyperspectral image classification. We present a couple graph based label propagation method, which contains both the adjacency graph and the similar graph. We propose to construct the similar graph by using the similar probability, which utilize the label similarity among examples probably. The adjacency graph was utilized by a common manifold learning method, which has effective improve the classification accuracy of hyperspectral data. The experiments indicate that the couple graph Laplacian which unite both the adjacency graph and the similar graph, produce superior classification results than other manifold Learning based graph Laplacian and Sparse representation based graph Laplacian in label propagation framework.

Beyond Learning by Doing: The Brain Compatible Approach.

ERIC Educational Resources Information Center

Roberts, Jay W.

2002-01-01

Principles of brain-based learning, including pattern and meaning making, parallel processing, and the role of stress and threat, are explained, along with their connections to longstanding practices of experiential education. The Brain Compatible Approach is one avenue for clarifying to mainstream educators how and why experiential methods are…

Room temperature synthesis of pH-switchable polyaniline quantum dots as a turn-on fluorescent probe for acidic biotarget labeling.

PubMed

Liu, Yanfeng; Ding, Yin; Gou, Huilin; Huang, Xin; Zhang, Guiyang; Zhang, Qi; Liu, Yunzhong; Meng, Zhen; Xi, Kai; Jia, Xudong

2018-04-05

The synthesis of well-defined light-element-derived quantum dots (LEQDs) with advanced optical properties under mild conditions is highly desirable yet challenging. Here, a polyaniline (PANI) structure is introduced into carbon-rich LEQDs to yield well-defined, fluorescent polyaniline quantum dots (PAQDs), PAQD24, through a one-pot room temperature reaction. The mild synthetic conditions effectively minimize the defects introduced during the conventional synthesis and endow PAQD24 with desirable optical properties, including a narrow emission band (full width at half maximum = 55 nm), an optimal quantum yield of 32.5% and two-photon fluorescence. Furthermore, the bandgap of PAQD24 is highly sensitive toward pH variations in the near-neutral region, due to the proton doping and dedoping of the PANI structure. Such unique properties together with its fine bio-compatibility enable the application of this material as a turn-on fluorescent probe for the labeling of acidic biotargets from sub-cellular to organ levels, providing potential applications in diagnosis and surgery guidance for certain diseases.

Stable isotope dimethyl labelling for quantitative proteomics and beyond

PubMed Central

Hsu, Jue-Liang; Chen, Shu-Hui

2016-01-01

Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644970

Mutual interferences and design principles for mechatronic devices in magnetic resonance imaging.

PubMed

Yu, Ningbo; Gassert, Roger; Riener, Robert

2011-07-01

Robotic and mechatronic devices that work compatibly with magnetic resonance imaging (MRI) are applied in diagnostic MRI, image-guided surgery, neurorehabilitation and neuroscience. MRI-compatible mechatronic systems must address the challenges imposed by the scanner's electromagnetic fields. We have developed objective quantitative evaluation criteria for device characteristics needed to formulate design guidelines that ensure MRI-compatibility based on safety, device functionality and image quality. The mutual interferences between an MRI system and mechatronic devices working in its vicinity are modeled and tested. For each interference, the involved components are listed, and a numerical measure for "MRI-compatibility" is proposed. These interferences are categorized into an MRI-compatibility matrix, with each element representing possible interactions between one part of the mechatronic system and one component of the electromagnetic fields. Based on this formulation, design principles for MRI-compatible mechatronic systems are proposed. Furthermore, test methods are developed to examine whether a mechatronic device indeed works without interferences within an MRI system. Finally, the proposed MRI-compatibility criteria and design guidelines have been applied to an actual design process that has been validated by the test procedures. Objective and quantitative MRI-compatibility measures for mechatronic and robotic devices have been established. Applying the proposed design principles, potential problems in safety, device functionality and image quality can be considered in the design phase to ensure that the mechatronic system will fulfill the MRI-compatibility criteria. New guidelines and test procedures for MRI instrument compatibility provide a rational basis for design and evaluation of mechatronic devices in various MRI applications. Designers can apply these criteria and use the tests, so that MRI-compatibility results can accrue to build an experiential database.

Research on segmentation based on multi-atlas in brain MR image

NASA Astrophysics Data System (ADS)

Qian, Yuejing

2018-03-01

Accurate segmentation of specific tissues in brain MR image can be effectively achieved with the multi-atlas-based segmentation method, and the accuracy mainly depends on the image registration accuracy and fusion scheme. This paper proposes an automatic segmentation method based on the multi-atlas for brain MR image. Firstly, to improve the registration accuracy in the area to be segmented, we employ a target-oriented image registration method for the refinement. Then In the label fusion, we proposed a new algorithm to detect the abnormal sparse patch and simultaneously abandon the corresponding abnormal sparse coefficients, this method is made based on the remaining sparse coefficients combined with the multipoint label estimator strategy. The performance of the proposed method was compared with those of the nonlocal patch-based label fusion method (Nonlocal-PBM), the sparse patch-based label fusion method (Sparse-PBM) and majority voting method (MV). Based on our experimental results, the proposed method is efficient in the brain MR images segmentation compared with MV, Nonlocal-PBM, and Sparse-PBM methods.

A Peptide-Based Method for 13C Metabolic Flux Analysis in Microbial Communities

PubMed Central

Ghosh, Amit; Nilmeier, Jerome; Weaver, Daniel; Adams, Paul D.; Keasling, Jay D.; Mukhopadhyay, Aindrila; Petzold, Christopher J.; Martín, Héctor García

2014-01-01

The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial communities is of crucial importance to understand and predict their behaviour. The most authoritative method of measuring intracellular fluxes, 13C Metabolic Flux Analysis (13C MFA), uses the labeling pattern obtained from metabolites (typically amino acids) during 13C labeling experiments to derive intracellular fluxes. However, these metabolite labeling patterns cannot easily be obtained for each of the members of the community. Here we propose a new type of 13C MFA that infers fluxes based on peptide labeling, instead of amino acid labeling. The advantage of this method resides in the fact that the peptide sequence can be used to identify the microbial species it originates from and, simultaneously, the peptide labeling can be used to infer intracellular metabolic fluxes. Peptide identity and labeling patterns can be obtained in a high-throughput manner from modern proteomics techniques. We show that, using this method, it is theoretically possible to recover intracellular metabolic fluxes in the same way as through the standard amino acid based 13C MFA, and quantify the amount of information lost as a consequence of using peptides instead of amino acids. We show that by using a relatively small number of peptides we can counter this information loss. We computationally tested this method with a well-characterized simple microbial community consisting of two species. PMID:25188426

SCFSLF-mediated cytosolic degradation of S-RNase is required for cross-pollen compatibility in S-RNase-based self-incompatibility in Petunia hybrida

PubMed Central

Liu, Wei; Fan, Jiangbo; Li, Junhui; Song, Yanzhai; Li, Qun; Zhang, Yu'e; Xue, Yongbiao

2014-01-01

Many flowering plants adopt self-incompatibility (SI) to maintain their genetic diversity. In species of Solanaceae, Plantaginaceae, and Rosaceae, SI is genetically controlled by a single S-locus with multiple haplotypes. The S-locus has been shown to encode S-RNases expressed in pistil and multiple SLF (S-locus F-box) proteins in pollen controlling the female and male specificity of SI, respectively. S-RNases appear to function as a cytotoxin to reject self-pollen. In addition, SLFs have been shown to form SCF (SKP1/Cullin1/F-box) complexes to serve as putative E3 ubiquitin ligase to interact with S-RNases. Previously, two different mechanisms, the S-RNase degradation and the S-RNase compartmentalization, have been proposed as the restriction mechanisms of S-RNase cytotoxicity allowing compatible pollination. In this study, we have provided several lines of evidence in support of the S-RNase degradation mechanism by a combination of cellular, biochemical and molecular biology approaches. First, both immunogold labeling and subcellular fractionation assays showed that two key pollen SI factors, PhS3L-SLF1 and PhSSK1 (SLF-interacting SKP1-like1) from Petunia hybrida, a Solanaceous species, are co-localized in cytosols of both pollen grains and tubes. Second, PhS3L-RNases are mainly detected in the cytosols of both self and non-self-pollen tubes after pollination. Third, we found that PhS-RNases selectively interact with PhSLFs by yeast two-hybrid and co-immunoprecipitation assays. Fourth, S-RNases are specifically degraded in compatible pollen tubes by non-self SLF action. Taken together, our results demonstrate that SCFSLF-mediated non-self S-RNase degradation occurs in the cytosol of pollen tube through the ubiquitin/26S proteasome system serving as the major mechanism to neutralize S-RNase cytotoxicity during compatible pollination in P. hybrida. PMID:25101113

Multi-Label Learning via Random Label Selection for Protein Subcellular Multi-Locations Prediction.

PubMed

Wang, Xiao; Li, Guo-Zheng

2013-03-12

Prediction of protein subcellular localization is an important but challenging problem, particularly when proteins may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular localization methods are only used to deal with the single-location proteins. In the past few years, only a few methods have been proposed to tackle proteins with multiple locations. However, they only adopt a simple strategy, that is, transforming the multi-location proteins to multiple proteins with single location, which doesn't take correlations among different subcellular locations into account. In this paper, a novel method named RALS (multi-label learning via RAndom Label Selection), is proposed to learn from multi-location proteins in an effective and efficient way. Through five-fold cross validation test on a benchmark dataset, we demonstrate our proposed method with consideration of label correlations obviously outperforms the baseline BR method without consideration of label correlations, indicating correlations among different subcellular locations really exist and contribute to improvement of prediction performance. Experimental results on two benchmark datasets also show that our proposed methods achieve significantly higher performance than some other state-of-the-art methods in predicting subcellular multi-locations of proteins. The prediction web server is available at http://levis.tongji.edu.cn:8080/bioinfo/MLPred-Euk/ for the public usage.

RJMCMC based Text Placement to Optimize Label Placement and Quantity

NASA Astrophysics Data System (ADS)

Touya, Guillaume; Chassin, Thibaud

2018-05-01

Label placement is a tedious task in map design, and its automation has long been a goal for researchers in cartography, but also in computational geometry. Methods that search for an optimal or nearly optimal solution that satisfies a set of constraints, such as label overlapping, have been proposed in the literature. Most of these methods mainly focus on finding the optimal position for a given set of labels, but rarely allow the removal of labels as part of the optimization. This paper proposes to apply an optimization technique called Reversible-Jump Markov Chain Monte Carlo that enables to easily model the removal or addition during the optimization iterations. The method, quite preliminary for now, is tested on a real dataset, and the first results are encouraging.

Al18F-Labeling Of Heat-Sensitive Biomolecules for Positron Emission Tomography Imaging

PubMed Central

Cleeren, Frederik; Lecina, Joan; Ahamed, Muneer; Raes, Geert; Devoogdt, Nick; Caveliers, Vicky; McQuade, Paul; Rubins, Daniel J; Li, Wenping; Verbruggen, Alfons; Xavier, Catarina; Bormans, Guy

2017-01-01

Positron emission tomography (PET) using radiolabeled biomolecules is a translational molecular imaging technology that is increasingly used in support of drug development. Current methods for radiolabeling biomolecules with fluorine-18 are laborious and require multistep procedures with moderate labeling yields. The Al18F-labeling strategy involves chelation in aqueous medium of aluminum mono[18F]fluoride ({Al18F}2+) by a suitable chelator conjugated to a biomolecule. However, the need for elevated temperatures (100-120 °C) required for the chelation reaction limits its widespread use. Therefore, we designed a new restrained complexing agent (RESCA) for application of the AlF strategy at room temperature. Methods. The new chelator RESCA was conjugated to three relevant biologicals and the constructs were labeled with {Al18F}2+ to evaluate the generic applicability of the one-step Al18F-RESCA-method. Results. We successfully labeled human serum albumin with excellent radiochemical yields in less than 30 minutes and confirmed in vivo stability of the Al18F-labeled protein in rats. In addition, we efficiently labeled nanobodies targeting the Kupffer cell marker CRIg, and performed µPET studies in healthy and CRIg deficient mice to demonstrate that the proposed radiolabeling method does not affect the functional integrity of the protein. Finally, an affibody targeting HER2 (PEP04314) was labeled site-specifically, and the distribution profile of (±)-[18F]AlF(RESCA)-PEP04314 in a rhesus monkey was compared with that of [18F]AlF(NOTA)-PEP04314 using whole-body PET/CT. Conclusion. This generic radiolabeling method has the potential to be a kit-based fluorine-18 labeling strategy, and could have a large impact on PET radiochemical space, potentially enabling the development of many new fluorine-18 labeled protein-based radiotracers. PMID:28824726

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Using Ensemble Decisions and Active Selection to Improve Low-Cost Labeling for Multi-View Data

NASA Technical Reports Server (NTRS)

Rebbapragada, Umaa; Wagstaff, Kiri L.

2011-01-01

This paper seeks to improve low-cost labeling in terms of training set reliability (the fraction of correctly labeled training items) and test set performance for multi-view learning methods. Co-training is a popular multiview learning method that combines high-confidence example selection with low-cost (self) labeling. However, co-training with certain base learning algorithms significantly reduces training set reliability, causing an associated drop in prediction accuracy. We propose the use of ensemble labeling to improve reliability in such cases. We also discuss and show promising results on combining low-cost ensemble labeling with active (low-confidence) example selection. We unify these example selection and labeling strategies under collaborative learning, a family of techniques for multi-view learning that we are developing for distributed, sensor-network environments.

Combination of diOlistic labeling with retrograde tract tracing and immunohistochemistry.

PubMed

Neely, M Diana; Stanwood, Gregg D; Deutch, Ariel Y

2009-11-15

Neuronal staining techniques have played a crucial role in the analysis of neuronal function. Several different staining techniques have been developed to allow morphological analyses of neurons. DiOlistic labeling, in which beads are coated with a lipophilic dye and then ballistically ejected onto brain tissue, has recently been introduced as a useful and simple means to label neurons and glia in their entirety. Although diOlistic labeling provides detailed information on the morphology of neurons, combining this approach with other staining methods is a significant advance. We have developed protocols that result in high quality diOlistically- and retrogradely-labeled or diOlistically-immunohistochemically labeled neurons. These dual-label methods require modification of fixation parameters and the restricted use of detergents for tissue permeabilization, and are readily applicable to a wide range of tracers and antibodies.

Combination of DiOlistic Labeling with Retrograde Tract Tracing and Immunohistochemistry

PubMed Central

Diana Neely, M.; Stanwood, Gregg D; Deutch, Ariel Y.

2009-01-01

Neuronal staining techniques have played a crucial role in the analysis of neuronal function. Several different staining techniques have been developed to allow morphological analyses of neurons. Recently diOlistic labeling, in which beads are coated with a lipophilic dye and then ballistically ejected onto brain tissue, has been developed as a useful and simple means to label neurons and glia in their entirety. Although diOlistic labeling provides detailed information on the morphology of neurons, combining this approach with other staining methods is a significant advance. We have developed protocols that result in high quality diOlistically- and retrogradely-labeled or diOlistically-immunohistochemically labeled neurons. These dual-label methods require modification of fixation parameters and the use of detergents for tissue permeabilization, and are readily applicable to a wide range of tracers and antibodies. PMID:19712695

Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

PubMed

Terada, Takaho; Yokoyama, Shigeyuki

2015-01-01

This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

PubMed

Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

2015-10-21

Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.

Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle.

PubMed

Britton, H G; Clarke, J B

1972-11-01

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of (32)P- and (14)C-labelled substrates at chemical equilibrium. 3. With (14)C-labelled substrates no induced transport was found over a wide concentration range, and with (32)P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. (14)C-labelled substrates exchange at twice the rate of (32)P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4x10(6)s(-1). The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the K(m) for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.

Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle

PubMed Central

Britton, H. G.; Clarke, J. B.

1972-01-01

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of 32P- and 14C-labelled substrates at chemical equilibrium. 3. With 14C-labelled substrates no induced transport was found over a wide concentration range, and with 32P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. 14C-labelled substrates exchange at twice the rate of 32P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4×106s−1. The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the Km for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase. PMID:4677138

Method for producing iron-based catalysts

DOEpatents

Farcasiu, Malvina; Kaufman, Phillip B.; Diehl, J. Rodney; Kathrein, Hendrik

1999-01-01

A method for preparing an acid catalyst having a long shelf-life is provided comprising doping crystalline iron oxides with lattice-compatible metals and heating the now-doped oxide with halogen compounds at elevated temperatures. The invention also provides for a catalyst comprising an iron oxide particle having a predetermined lattice structure, one or more metal dopants for said iron oxide, said dopants having an ionic radius compatible with said lattice structure; and a halogen bound with the iron and the metal dopants on the surface of the particle.

Flammability, Offgassing, and Compatibility Requirements and Test Procedures. Interim NASA Technical Standard

NASA Technical Reports Server (NTRS)

2009-01-01

This Interim Standard establishes requirements for evaluation, testing, and selection of materials that are intended for use in space vehicles, associated Ground Support Equipment (GSE), and facilities used during assembly, test, and flight operations. Included are requirements, criteria, and test methods for evaluating the flammability, offgassing, and compatibility of materials.

A Laboratory Exercise for Compatibility Testing of Hazardous Wastes in an Environmental Analysis Course.

ERIC Educational Resources Information Center

Chang, J. C.; And Others

1986-01-01

Discusses a new program at the University of Michigan in hazardous waste management. Describes a laboratory demonstration that deals with the reactivity and potential violence of several reactions that may be encountered on a hazardous waste site. Provides criteria for selecting particular compatibility testing methods. (TW)

A third option for climate policy within potential limits to growth

NASA Astrophysics Data System (ADS)

van den Bergh, Jeroen C. J. M.

2017-02-01

Climate change has revived debates around the concept of limits to growth, 45 years after it was first proposed. Many citizens, scientists and politicians fear that stringent climate policy will harm economic growth. Some are anti-growth, whereas others believe green growth is compatible with a transition to a low-carbon economy. As the window to curb warming at 2 °C closes, this debate will intensify. This Review critically reflects on both positions, providing an overview of existing literature on the growth versus climate debate. Both positions are argued here to jeopardize environmental or social goals. A third position, labelled an 'agrowth' strategy, is proposed to depolarize the debate and reduce resistance to climate policies.

Modular, Antibody-free Time-Resolved LRET Kinase Assay Enabled by Quantum Dots and Tb3+-sensitizing Peptides

NASA Astrophysics Data System (ADS)

Cui, Wei; Parker, Laurie L.

2016-07-01

Fluorescent drug screening assays are essential for tyrosine kinase inhibitor discovery. Here we demonstrate a flexible, antibody-free TR-LRET kinase assay strategy that is enabled by the combination of streptavidin-coated quantum dot (QD) acceptors and biotinylated, Tb3+ sensitizing peptide donors. By exploiting the spectral features of Tb3+ and QD, and the high binding affinity of the streptavidin-biotin interaction, we achieved multiplexed detection of kinase activity in a modular fashion without requiring additional covalent labeling of each peptide substrate. This strategy is compatible with high-throughput screening, and should be adaptable to the rapidly changing workflows and targets involved in kinase inhibitor discovery.

Portion Size Labeling and Intended Soft Drink Consumption: The Impact of Labeling Format and Size Portfolio

ERIC Educational Resources Information Center

Vermeer, Willemijn M.; Steenhuis, Ingrid H. M.; Leeuwis, Franca H.; Bos, Arjan E. R.; de Boer, Michiel; Seidell, Jacob C.

2010-01-01

Objective: To assess what portion size labeling "format" is most promising in helping consumers selecting appropriate soft drink sizes, and whether labeling impact depends on the size portfolio. Methods: An experimental study was conducted in fast-food restaurants in which 2 labeling formats (ie, reference portion size and small/medium/large…

Methyl-coenzyme M reductase from methanogenic archaea: isotope effects on label exchange and ethane formation with the homologous substrate ethyl-coenzyme M.

PubMed

Scheller, Silvan; Goenrich, Meike; Thauer, Rudolf K; Jaun, Bernhard

2013-10-09

Ethyl-coenzyme M (CH3CH2-S-CH2CH2-SO3(-), Et-S-CoM) serves as a homologous substrate for the enzyme methyl-coenzyme M reductase (MCR) resulting in the product ethane instead of methane. The catalytic reaction proceeds via an intermediate that already contains all six C-H bonds of the product. Because product release occurs after a second, rate-limiting step, many cycles of intermediate formation and reconversion to substrate occur before a substantial amount of ethane is released. In deuterated buffer, the intermediate becomes labeled, and C-H activation in the back reaction rapidly leads to labeled Et-S-CoM, which enables intermediate formation to be detected. Here, we present a comprehensive analysis of this pre-equilibrium. (2)H- and (13)C-labeled isotopologues of Et-S-CoM were used as the substrates, and the time course of each isotopologue was followed by NMR spectroscopy. A kinetic simulation including kinetic isotope effects allowed determination of the primary and α- and β-secondary isotope effects for intermediate formation and for the C-H/C-D bond activation in the ethane-containing intermediate. The values obtained are in accordance with those found for the native substrate Me-S-CoM (see preceding publication, Scheller, S.; Goenrich, M.; Thauer, R. K.; Jaun, B. J. Am. Chem. Soc. 2013, 135, DOI: 10.1021/ja406485z) and thus imply the same catalytic mechanism for both substrates. The experiment by Floss and co-workers, demonstrating a net inversion of configuration to chiral ethane with CH3CDT-S-CoM as the substrate, is compatible with the observed rapid isotope exchange if the isotope effects measured here are taken into account.

Proximal Bright Vessel Sign on Arterial Spin Labeling Magnetic Resonance Imaging in Acute Cardioembolic Cerebral Infarction.

PubMed

Kato, Ayumi; Shinohara, Yuki; Kuya, Keita; Sakamoto, Makoto; Kowa, Hisanori; Ogawa, Toshihide

2017-07-01

The congestion of spin-labeled blood at large-vessel occlusion can present as hyperintense signals on perfusion magnetic resonance imaging with 3-dimensional pseudo-continuous arterial spin labeling (proximal bright vessel sign). The purpose of this study was to clarify the difference between proximal bright vessel sign and susceptibility vessel sign in acute cardioembolic cerebral infarction. Forty-two patients with cardioembolic cerebral infarction in the anterior circulation territory underwent magnetic resonance imaging including diffusion-weighted imaging, 3-dimensional pseudo-continuous arterial spin labeling perfusion magnetic resonance imaging, T2*-weighted imaging, and 3-dimensional time-of-flight magnetic resonance angiography using a 3-T magnetic resonance scanner. Visual assessments of proximal bright vessel sign and the susceptibility vessel sign were performed by consensus of 2 experienced neuroradiologists. The relationship between these signs and the occlusion site of magnetic resonance angiography was also investigated. Among 42 patients with cardioembolic cerebral infarction, 24 patients showed proximal bright vessel sign (57.1%) and 25 showed susceptibility vessel sign (59.5%). There were 19 cases of proximal bright vessel sign and susceptibility vessel sign-clear, 12 cases of proximal bright vessel sign and susceptibility vessel sign-unclear, and 11 mismatched cases. Four out of 6 patients with proximal bright vessel sign-unclear and susceptibility vessel sign-clear showed distal middle cerebral artery occlusion, and 2 out of 5 patients with proximal bright vessel sign-clear and susceptibility vessel sign-unclear showed no occlusion on magnetic resonance angiography. Proximal bright vessel sign is almost compatible with susceptibility vessel sign in patients with cardioembolic cerebral infarction. Copyright © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Introducing Euro-Glo, a rare earth metal chelate with numerous applications for the fluorescent localization of myelin and amyloid plaques in brain tissue sections.

PubMed

Schmued, Larry; Raymick, James

2017-03-01

The vast majority of fluorochromes are organic in nature and none of the few existing chelates have been applied as histological tracers for localizing brain anatomy and pathology. In this study we have developed and characterized a Europium chelate with the ability to fluorescently label normal and pathological myelin in control and toxicant-exposed rats, as well as the amyloid plaques in aged AD/Tg mice. This study demonstrates how Euro-Glo can be used for the detailed labeling of both normal myelination in the control rat as well as myelin pathology in the kainic acid exposed rat. In addition, this study demonstrates how E-G will label the shell of amyloid plaques in an AD/Tg mouse model of Alzheimer's disease a red color, while the plaque core appears blue in color. The observed E-G staining pattern is compared with that of well characterized tracers specific for the localization of myelin (Black-Gold II), degenerating neurons (Fluoro-Jade C), A-beta aggregates (Amylo-Glo) and glycolipids (PAS). This study represents the first time a rare earth metal (REM) chelate has been used as a histochemical tracer in the brain. This novel tracer, Euro-Glo (E-G), exhibits numerous advantages over conventional organic fluorophores including high intensity emission, high resistance to fading, compatibility with multiple labeling protocols, high Stoke's shift value and an absence of bleed-through of the signal through other filters. Euro-Glo represents the first fluorescent metal chelate to be used as a histochemical tracer, specifically to localize normal and pathological myelin as well as amyloid plaques. Copyright © 2016. Published by Elsevier B.V.

Biocompatible water soluble quantum dots as new biophotonic tools for hematologic cells: applications for flow cell cytometry

NASA Astrophysics Data System (ADS)

Lira, Rafael B.; de Sales Neto, Antonio T.; Carvalho, Kilmara K. H. G.; Leite, Elisa S.; Brasil, Aluizio G., Jr.; Azevedo, Denise P. L.; Cabral Filho, Paulo E.; Cavalcanti, Mariana B.; Amaral, Ademir J.; Farias, Patricía M. A.; Santos, Beate S.; Fontes, Adriana

2010-02-01

Quantum dots (QDs) are a promising class of fluorescent probes that can be conjugated to a variety of specific cell antibodies. For this reason, simple, cheap and reproducible routes of QDśs syntheses are the main goal of many researches in this field. The main objective of this work was to demonstrate the ability of QDs as biolabels for flow cell cytometry analysis. We have synthesized biocompatible water soluble CdS/Cd(OH)2 and CdTe/CdS QDs and applied them as fluorescent labels of hematologic cells. CdTe/CdS QDs was prepared using using a simple aqueous route with mercaptoacetic acid and mercaptopropionic acid as stabilizing agents. The resulting CdTe/CdS QDs can target biological membrane proteins and can also be internalized by cells. We applied the CdTe/CdS QDs as biolabels of human lymphocytes and compared the results obtained for lymphocytes treated and non-treated with permeabilizing agents for cell membranes. Permeabilized cells present higher fluorescence pattern than non permeabilized ones. We associated antibody A to the CdS/Cd(OH)2 QDs to label type A red blood cell (RBC). In this case, the O erythrocytes were used as the negative control. The results demonstrate that QDs were successfully functionalized with antibody A. There was a specific binding of QDs-antibody A to RBC membrane antigen only for A RBCs. We have also monitored QDs-hematologic cell interaction by using fluorescence microscopy. Our method shows that QDs can be conjugated to a variety of specific cell antibodies and can become a potential, highly efficient and low cost diagnostic tool for flow cell cytometry, very compatible with the lasers and filters used in this kind of equipments.

Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

2016-05-10

Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

Development and utilization of a web-based application as a robust radiology teaching tool (radstax) for medical student anatomy teaching.

PubMed

Colucci, Philip G; Kostandy, Petro; Shrauner, William R; Arleo, Elizabeth; Fuortes, Michele; Griffin, Andrew S; Huang, Yun-Han; Juluru, Krishna; Tsiouris, Apostolos John

2015-02-01

Rationale and Objectives: The primary role of radiology in the preclinical setting is the use of imaging to improve students' understanding of anatomy. Many currently available Web-based anatomy programs include either suboptimal or overwhelming levels of detail for medical students.Our objective was to develop a user-friendly software program that anatomy instructors can completely tailor to match the desired level of detail for their curriculum, meets the unique needs of the first- and the second-year medical students, and is compatible with most Internet browsers and tablets.Materials and Methods: RadStax is a Web-based application developed using free, open-source, ubiquitous software. RadStax was first introduced as an interactive resource for independent study and later incorporated into lectures. First- and second-year medical students were surveyed for quantitative feedback regarding their experience.Results: RadStax was successfully introduced into our medical school curriculum. It allows the creation of learning modules with labeled multiplanar (MPR) image sets, basic anatomic information, and a self-assessment feature. The program received overwhelmingly positive feedback from students. Of 115 students surveyed, 87.0% found it highly effective as a study tool and 85.2% reported high user satisfaction with the program.Conclusions: RadStax is a novel application for instructors wishing to create an atlas of labeled MPR radiologic studies tailored to meet the specific needs their curriculum. Simple and focused, it provides an interactive experience for students similar to the practice of radiologists.This program is a robust anatomy teaching tool that effectively aids in educating the preclinical medical student.

Development and Utilization of a Web-Based Application as a Robust Radiology Teaching Tool (RadStax) for Medical Student Anatomy Teaching

PubMed Central

Colucci, Philip G.; Kostandy, Petro; Shrauner, William R.; Arleo, Elizabeth; Fuortes, Michele; Griffin, Andrew S.; Huang, Yun-Han; Juluru, Krishna; Tsiouris, Apostolos John

2016-01-01

Rationale and Objectives The primary role of radiology in the preclinical setting is the use of imaging to improve students’ understanding of anatomy. Many currently available Web-based anatomy programs include either suboptimal or overwhelming levels of detail for medical students. Our objective was to develop a user-friendly software program that anatomy instructors can completely tailor to match the desired level of detail for their curriculum, meets the unique needs of the first- and the second-year medical students, and is compatible with most Internet browsers and tablets. Materials and Methods RadStax is a Web-based application developed using free, open-source, ubiquitous software. RadStax was first introduced as an interactive resource for independent study and later incorporated into lectures. First- and second-year medical students were surveyed for quantitative feedback regarding their experience. Results RadStax was successfully introduced into our medical school curriculum. It allows the creation of learning modules with labeled multiplanar (MPR) image sets, basic anatomic information, and a self-assessment feature. The program received overwhelmingly positive feedback from students. Of 115 students surveyed, 87.0% found it highly effective as a study tool and 85.2% reported high user satisfaction with the program. Conclusions RadStax is a novel application for instructors wishing to create an atlas of labeled MPR radiologic studies tailored to meet the specific needs their curriculum. Simple and focused, it provides an interactive experience for students similar to the practice of radiologists. This program is a robust anatomy teaching tool that effectively aids in educating the preclinical medical student. PMID:25964956

Risk assessment and quality improvement of liquid waste management in Taiwan University chemical laboratories.

PubMed

Ho, Chao-Chung; Chen, Ming-Shu

2018-01-01

The policy of establishing new universities across Taiwan has led to an increase in the number of universities, and many schools have constructed new laboratories to meet students' academic needs. In recent years, there has been an increase in the number of laboratory accidents from the liquid waste in universities. Therefore, how to build a safety system for laboratory liquid waste disposal has become an important issue in the environmental protection, safety, and hygiene of all universities. This study identifies the risk factors of liquid waste disposal and presents an agenda for practices to laboratory managers. An expert questionnaire is adopted to probe into the risk priority procedures of liquid waste disposal; then, the fuzzy theory-based FMEA method and the traditional FMEA method are employed to analyze and improve the procedures for liquid waste disposal. According to the research results, the fuzzy FMEA method is the most effective, and the top 10 potential disabling factors are prioritized for improvement according to the risk priority number (RNP), including "Unclear classification", "Gathering liquid waste without a funnel or a drain pan", "Lack of a clearance and transport contract", "Liquid waste spill during delivery", "Spill over", "Decentralized storage", "Calculating weight in the wrong way", "Compatibility between the container material and the liquid waste", "Lack of dumping and disposal tools", and "Lack of a clear labels for liquid waste containers". After tracking improvements, the overall improvement rate rose to 60.2%. Copyright © 2017 Elsevier Ltd. All rights reserved.

Radio-metabolite analysis of carbon-11 biochemical partitioning to non-structural carbohydrates for integrated metabolism and transport studies.

PubMed

Babst, Benjamin A; Karve, Abhijit A; Judt, Tatjana

2013-06-01

Metabolism and phloem transport of carbohydrates are interactive processes, yet each is often studied in isolation from the other. Carbon-11 ((11)C) has been successfully used to study transport and allocation processes dynamically over time. There is a need for techniques to determine metabolic partitioning of newly fixed carbon that are compatible with existing non-invasive (11)C-based methodologies for the study of phloem transport. In this report, we present methods using (11)C-labeled CO2 to trace carbon partitioning to the major non-structural carbohydrates in leaves-sucrose, glucose, fructose and starch. High-performance thin-layer chromatography (HPTLC) was adapted to provide multisample throughput, raising the possibility of measuring different tissues of the same individual plant, or for screening multiple plants. An additional advantage of HPTLC was that phosphor plate imaging of radioactivity had a much higher sensitivity and broader range of sensitivity than radio-HPLC detection, allowing measurement of (11)C partitioning to starch, which was previously not possible. Because of the high specific activity of (11)C and high sensitivity of detection, our method may have additional applications in the study of rapid metabolic responses to environmental changes that occur on a time scale of minutes. The use of this method in tandem with other (11)C assays for transport dynamics and whole-plant partitioning makes a powerful combination of tools to study carbohydrate metabolism and whole-plant transport as integrated processes.

Ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration.

PubMed

Mort, Richard L; Keighren, Margaret; Hay, Leonard; Jackson, Ian J

2014-05-19

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles(1,2). Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture(3-6). Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue(3,6). High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture(6). By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.

An algorithm for optimal fusion of atlases with different labeling protocols

PubMed Central

Iglesias, Juan Eugenio; Sabuncu, Mert Rory; Aganj, Iman; Bhatt, Priyanka; Casillas, Christen; Salat, David; Boxer, Adam; Fischl, Bruce; Van Leemput, Koen

2014-01-01

In this paper we present a novel label fusion algorithm suited for scenarios in which different manual delineation protocols with potentially disparate structures have been used to annotate the training scans (hereafter referred to as “atlases”). Such scenarios arise when atlases have missing structures, when they have been labeled with different levels of detail, or when they have been taken from different heterogeneous databases. The proposed algorithm can be used to automatically label a novel scan with any of the protocols from the training data. Further, it enables us to generate new labels that are not present in any delineation protocol by defining intersections on the underling labels. We first use probabilistic models of label fusion to generalize three popular label fusion techniques to the multi-protocol setting: majority voting, semi-locally weighted voting and STAPLE. Then, we identify some shortcomings of the generalized methods, namely the inability to produce meaningful posterior probabilities for the different labels (majority voting, semi-locally weighted voting) and to exploit the similarities between the atlases (all three methods). Finally, we propose a novel generative label fusion model that can overcome these drawbacks. We use the proposed method to combine four brain MRI datasets labeled with different protocols (with a total of 102 unique labeled structures) to produce segmentations of 148 brain regions. Using cross-validation, we show that the proposed algorithm outperforms the generalizations of majority voting, semi-locally weighted voting and STAPLE (mean Dice score 83%, vs. 77%, 80% and 79%, respectively). We also evaluated the proposed algorithm in an aging study, successfully reproducing some well-known results in cortical and subcortical structures. PMID:25463466

An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

PubMed

Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Shoffner, Matthew; Albers, Aaron E; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

2015-11-19

Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins.

Preparation, characterization and pharmacokinetics of fluorescence labeled propylene glycol alginate sodium sulfate

NASA Astrophysics Data System (ADS)

Li, Pengli; Li, Chunxia; Xue, Yiting; Zhang, Yang; Liu, Hongbing; Zhao, Xia; Yu, Guangli; Guan, Huashi

2014-08-01

A rapid and sensitive fluorescence labeling method was developed and validated for the microanalysis of a sulfated polysaccharide drug,namely propylene glycol alginate sodium sulfate (PSS), in rat plasma. Fluorescein isothiocyanate (FITC) was selected to label PSS, and 1, 6-diaminohexane was used to link PSS and FITC in order to prepare FITC-labeled PSS (F-PSS) through a reductive amination reaction. F-PSS was identified by UV-Vis, FT-IR and 1H-NMR spectrum. The cell stability and cytotoxicity of F-PSS were tested in Madin-Darby canine kidney (MDCK) cells. The results indicated that the labeling efficiency of F-PSS was 0.522% ± 0.0248% and the absolute bioavailability was 8.39%. F-PSS was stable in MDCK cells without obvious cytotoxicity. The method was sensitive and reliable; it showed a good linearity, precision, recovery and stability. The FITC labeling method can be applied to investigating the absorption and metabolism of PSS and other polysaccharides in biological samples.

Development And Evaluation Of Stable Isotope And Fluorescent Labeling And Detection Methodologies For Tracking Injected Bacteria During In Situ Bioremediation

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

Mark E. Fuller; Tullis C. Onstott

2003-12-17

This report summarizes the results of a research project conducted to develop new methods to label bacterial cells so that they could be tracked and enumerated as they move in the subsurface after they are introduced into the groundwater (i.e., during bioaugmentation). Labeling methods based on stable isotopes of carbon (13C) and vital fluorescent stains were developed. Both approaches proved successful with regards to the ability to effectively label bacterial cells. Several methods for enumeration of fluorescently-labeled cells were developed and validated, including near-real time microplate spectrofluorometry that could be performed in the field. However, the development of a novelmore » enumeration method for the 13C-enriched cells, chemical reaction interface/mass spectrometry (CRIMS), was not successful due to difficulties with the proposed instrumentation. Both labeling methodologies were successfully evaluated and validated during laboratory- and field-scale bacterial transport experiments. The methods developed during this research should be useful for future bacterial transport work as well as other microbial ecology research in a variety of environments. A full bibliography of research articles and meeting presentations related to this project is included (including web links to abstracts and full text reprints).« less

Comparing model-based and model-free analysis methods for QUASAR arterial spin labeling perfusion quantification.

PubMed

Chappell, Michael A; Woolrich, Mark W; Petersen, Esben T; Golay, Xavier; Payne, Stephen J

2013-05-01

Amongst the various implementations of arterial spin labeling MRI methods for quantifying cerebral perfusion, the QUASAR method is unique. By using a combination of labeling with and without flow suppression gradients, the QUASAR method offers the separation of macrovascular and tissue signals. This permits local arterial input functions to be defined and "model-free" analysis, using numerical deconvolution, to be used. However, it remains unclear whether arterial spin labeling data are best treated using model-free or model-based analysis. This work provides a critical comparison of these two approaches for QUASAR arterial spin labeling in the healthy brain. An existing two-component (arterial and tissue) model was extended to the mixed flow suppression scheme of QUASAR to provide an optimal model-based analysis. The model-based analysis was extended to incorporate dispersion of the labeled bolus, generally regarded as the major source of discrepancy between the two analysis approaches. Model-free and model-based analyses were compared for perfusion quantification including absolute measurements, uncertainty estimation, and spatial variation in cerebral blood flow estimates. Major sources of discrepancies between model-free and model-based analysis were attributed to the effects of dispersion and the degree to which the two methods can separate macrovascular and tissue signal. Copyright © 2012 Wiley Periodicals, Inc.

Manifold Regularized Experimental Design for Active Learning.

PubMed

Zhang, Lining; Shum, Hubert P H; Shao, Ling

2016-12-02

Various machine learning and data mining tasks in classification require abundant data samples to be labeled for training. Conventional active learning methods aim at labeling the most informative samples for alleviating the labor of the user. Many previous studies in active learning select one sample after another in a greedy manner. However, this is not very effective because the classification models has to be retrained for each newly labeled sample. Moreover, many popular active learning approaches utilize the most uncertain samples by leveraging the classification hyperplane of the classifier, which is not appropriate since the classification hyperplane is inaccurate when the training data are small-sized. The problem of insufficient training data in real-world systems limits the potential applications of these approaches. This paper presents a novel method of active learning called manifold regularized experimental design (MRED), which can label multiple informative samples at one time for training. In addition, MRED gives an explicit geometric explanation for the selected samples to be labeled by the user. Different from existing active learning methods, our method avoids the intrinsic problems caused by insufficiently labeled samples in real-world applications. Various experiments on synthetic datasets, the Yale face database and the Corel image database have been carried out to show how MRED outperforms existing methods.

End labeling procedures: an overview.

PubMed

Hilario, Elena

2004-09-01

There are two ways to label a DNA molecular; by the ends or all along the molecule. End labeling can be performed at the 3'- or 5'-end. Labeling at the 3' end is performed by filling 3'-end recessed ends with a mixture or labeled and unlabeled dNTPs using Klenow or T4 DNA polymerases. Both reactions are template dependent. Terminal deoxynucleotide transferase incorporates dNTPs at the 3' end of any kind of DNA molecule or RNA. Labels incorporated at the 3'-end of the DNA molecule prevent any further extension or ligation to any other molecule, but this can be overcome by labeling the 5'-end of the desired DNA molecule. 5'-end labeling is performed by enzymatic methods (T4 polynucleotide kinase exchange and forward reactions), by chemical modification of sensitized oligonucleotides with phosphoroamidite, or by combined methods. Probe cleanup is recommended when high background problems occur, but caution should be taken not to damage the attached probe with harsh chemicals or by light exposure.

Quadrature imposition of compatibility conditions in Chebyshev methods

NASA Technical Reports Server (NTRS)

Gottlieb, D.; Streett, C. L.

1990-01-01

Often, in solving an elliptic equation with Neumann boundary conditions, a compatibility condition has to be imposed for well-posedness. This condition involves integrals of the forcing function. When pseudospectral Chebyshev methods are used to discretize the partial differential equation, these integrals have to be approximated by an appropriate quadrature formula. The Gauss-Chebyshev (or any variant of it, like the Gauss-Lobatto) formula can not be used here since the integrals under consideration do not include the weight function. A natural candidate to be used in approximating the integrals is the Clenshaw-Curtis formula, however it is shown that this is the wrong choice and it may lead to divergence if time dependent methods are used to march the solution to steady state. The correct quadrature formula is developed for these problems. This formula takes into account the degree of the polynomials involved. It is shown that this formula leads to a well conditioned Chebyshev approximation to the differential equations and that the compatibility condition is automatically satisfied.

Rate-Compatible LDPC Codes with Linear Minimum Distance

NASA Technical Reports Server (NTRS)

Divsalar, Dariush; Jones, Christopher; Dolinar, Samuel

2009-01-01

A recently developed method of constructing protograph-based low-density parity-check (LDPC) codes provides for low iterative decoding thresholds and minimum distances proportional to block sizes, and can be used for various code rates. A code constructed by this method can have either fixed input block size or fixed output block size and, in either case, provides rate compatibility. The method comprises two submethods: one for fixed input block size and one for fixed output block size. The first mentioned submethod is useful for applications in which there are requirements for rate-compatible codes that have fixed input block sizes. These are codes in which only the numbers of parity bits are allowed to vary. The fixed-output-blocksize submethod is useful for applications in which framing constraints are imposed on the physical layers of affected communication systems. An example of such a system is one that conforms to one of many new wireless-communication standards that involve the use of orthogonal frequency-division modulation

A new dimethyl labeling-based SID-MRM-MS method and its application to three proteases involved in insulin maturation.

PubMed

Cheng, Dongwan; Zheng, Li; Hou, Junjie; Wang, Jifeng; Xue, Peng; Yang, Fuquan; Xu, Tao

2015-01-01

The absolute quantification of target proteins in proteomics involves stable isotope dilution coupled with multiple reactions monitoring mass spectrometry (SID-MRM-MS). The successful preparation of stable isotope-labeled internal standard peptides is an important prerequisite for the SID-MRM absolute quantification methods. Dimethyl labeling has been widely used in relative quantitative proteomics and it is fast, simple, reliable, cost-effective, and applicable to any protein sample, making it an ideal candidate method for the preparation of stable isotope-labeled internal standards. MRM mass spectrometry is of high sensitivity, specificity, and throughput characteristics and can quantify multiple proteins simultaneously, including low-abundance proteins in precious samples such as pancreatic islets. In this study, a new method for the absolute quantification of three proteases involved in insulin maturation, namely PC1/3, PC2 and CPE, was developed by coupling a stable isotope dimethyl labeling strategy for internal standard peptide preparation with SID-MRM-MS quantitative technology. This method offers a new and effective approach for deep understanding of the functional status of pancreatic β cells and pathogenesis in diabetes.

A Cluster-then-label Semi-supervised Learning Approach for Pathology Image Classification.

PubMed

Peikari, Mohammad; Salama, Sherine; Nofech-Mozes, Sharon; Martel, Anne L

2018-05-08

Completely labeled pathology datasets are often challenging and time-consuming to obtain. Semi-supervised learning (SSL) methods are able to learn from fewer labeled data points with the help of a large number of unlabeled data points. In this paper, we investigated the possibility of using clustering analysis to identify the underlying structure of the data space for SSL. A cluster-then-label method was proposed to identify high-density regions in the data space which were then used to help a supervised SVM in finding the decision boundary. We have compared our method with other supervised and semi-supervised state-of-the-art techniques using two different classification tasks applied to breast pathology datasets. We found that compared with other state-of-the-art supervised and semi-supervised methods, our SSL method is able to improve classification performance when a limited number of labeled data instances are made available. We also showed that it is important to examine the underlying distribution of the data space before applying SSL techniques to ensure semi-supervised learning assumptions are not violated by the data.

Active learning based segmentation of Crohns disease from abdominal MRI.

PubMed

Mahapatra, Dwarikanath; Vos, Franciscus M; Buhmann, Joachim M

2016-05-01

This paper proposes a novel active learning (AL) framework, and combines it with semi supervised learning (SSL) for segmenting Crohns disease (CD) tissues from abdominal magnetic resonance (MR) images. Robust fully supervised learning (FSL) based classifiers require lots of labeled data of different disease severities. Obtaining such data is time consuming and requires considerable expertise. SSL methods use a few labeled samples, and leverage the information from many unlabeled samples to train an accurate classifier. AL queries labels of most informative samples and maximizes gain from the labeling effort. Our primary contribution is in designing a query strategy that combines novel context information with classification uncertainty and feature similarity. Combining SSL and AL gives a robust segmentation method that: (1) optimally uses few labeled samples and many unlabeled samples; and (2) requires lower training time. Experimental results show our method achieves higher segmentation accuracy than FSL methods with fewer samples and reduced training effort. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Automated identification of cone photoreceptors in adaptive optics retinal images.

PubMed

Li, Kaccie Y; Roorda, Austin

2007-05-01

In making noninvasive measurements of the human cone mosaic, the task of labeling each individual cone is unavoidable. Manual labeling is a time-consuming process, setting the motivation for the development of an automated method. An automated algorithm for labeling cones in adaptive optics (AO) retinal images is implemented and tested on real data. The optical fiber properties of cones aided the design of the algorithm. Out of 2153 manually labeled cones from six different images, the automated method correctly identified 94.1% of them. The agreement between the automated and the manual labeling methods varied from 92.7% to 96.2% across the six images. Results between the two methods disagreed for 1.2% to 9.1% of the cones. Voronoi analysis of large montages of AO retinal images confirmed the general hexagonal-packing structure of retinal cones as well as the general cone density variability across portions of the retina. The consistency of our measurements demonstrates the reliability and practicality of having an automated solution to this problem.

An Upgrade Pinning Block: A Mechanical Practical Aid for Fast Labelling of the Insect Specimens.

PubMed

Ghafouri Moghaddam, Mohammad Hossein; Ghafouri Moghaddam, Mostafa; Rakhshani, Ehsan; Mokhtari, Azizollah

2017-01-01

A new mechanical innovation is described to deal with standard labelling of dried specimens on triangular cards and/or pinned specimens in personal and public collections. It works quickly, precisely, and easily and is very useful for maintaining label uniformity in collections. The tools accurately sets the position of labels in the shortest possible time. This tools has advantages including rapid processing, cost effectiveness, light weight, and high accuracy, compared to conventional methods. It is fully customisable, compact, and does not require specialist equipment to assemble. Conventional methods generally require locating holes on the pinning block surface when labelling with a resulting risk to damage of the specimens. Insects of different orders can be labelled by this simple and effective tool.

An Upgrade Pinning Block: A Mechanical Practical Aid for Fast Labelling of the Insect Specimens

PubMed Central

Ghafouri Moghaddam, Mohammad Hossein; Rakhshani, Ehsan; Mokhtari, Azizollah

2017-01-01

Abstract A new mechanical innovation is described to deal with standard labelling of dried specimens on triangular cards and/or pinned specimens in personal and public collections. It works quickly, precisely, and easily and is very useful for maintaining label uniformity in collections. The tools accurately sets the position of labels in the shortest possible time. This tools has advantages including rapid processing, cost effectiveness, light weight, and high accuracy, compared to conventional methods. It is fully customisable, compact, and does not require specialist equipment to assemble. Conventional methods generally require locating holes on the pinning block surface when labelling with a resulting risk to damage of the specimens. Insects of different orders can be labelled by this simple and effective tool. PMID:29104440

Prototype of a computer method for designing and analyzing heating, ventilating and air conditioning proportional, electronic control systems

NASA Astrophysics Data System (ADS)

Barlow, Steven J.

1986-09-01

The Air Force needs a better method of designing new and retrofit heating, ventilating and air conditioning (HVAC) control systems. Air Force engineers currently use manual design/predict/verify procedures taught at the Air Force Institute of Technology, School of Civil Engineering, HVAC Control Systems course. These existing manual procedures are iterative and time-consuming. The objectives of this research were to: (1) Locate and, if necessary, modify an existing computer-based method for designing and analyzing HVAC control systems that is compatible with the HVAC Control Systems manual procedures, or (2) Develop a new computer-based method of designing and analyzing HVAC control systems that is compatible with the existing manual procedures. Five existing computer packages were investigated in accordance with the first objective: MODSIM (for modular simulation), HVACSIM (for HVAC simulation), TRNSYS (for transient system simulation), BLAST (for building load and system thermodynamics) and Elite Building Energy Analysis Program. None were found to be compatible or adaptable to the existing manual procedures, and consequently, a prototype of a new computer method was developed in accordance with the second research objective.

A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells

PubMed Central

Robinet, Peggy; Wang, Zeneng; Hazen, Stanley L.; Smith, Jonathan D.

2010-01-01

A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells. PMID:20688754

A versatile new technique to clear mouse and human brain

NASA Astrophysics Data System (ADS)

Costantini, Irene; Di Giovanna, Antonino Paolo; Allegra Mascaro, Anna Letizia; Silvestri, Ludovico; Müllenbroich, Marie Caroline; Sacconi, Leonardo; Pavone, Francesco S.

2015-07-01

Large volumes imaging with microscopic resolution is limited by light scattering. In the last few years based on refractive index matching, different clearing approaches have been developed. Organic solvents and water-based optical clearing agents have been used for optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a highly expensive refractive index matching solution limiting practical applicability. In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2'-thiodiethanol (TDE) to clear mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for CLARITY. The potential of this method has been explored by imaging a whole transgenic mouse brain with the light sheet microscope. Moreover we apply this technique also on blocks of dysplastic human brain tissue transformed with CLARITY and labeled with different antibody. This clearing approach significantly expands the application of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in mouse and human brain.

A simple method for comparing immunogold distributions in two or more experimental groups illustrated using GLUT1 labelling of isolated trophoblast cells.

PubMed

Mayhew, T M; Desoye, G

2004-07-01

Colloidal gold-labelling, combined with transmission electron microscopy, is a valuable technique for high-resolution immunolocalization of identified antigens in different subcellular compartments. Whilst the technique has been applied to placental tissues, few quantitative studies have been made. Subcellular compartments exist in three main categories (viz. organelles, membranes, filaments/tubules) and this affects the possibilities for quantification. Generally, gold particles are counted in order to compare either (a) compartments within an experimental group or (b) compartmental labelling distributions between groups. For the former, recent developments make it possible to test whether or not there is differential (nonrandom) labelling of compartments. The methods (relative labelling index and labelling density) are ideally suited to analysing label in one category of compartment (organelle or membrane or filament) but may be adapted to deal with a mixture of categories. They also require information about compartment size (e.g. profile area or trace length). Here, a simple and efficient method for drawing between-group comparisons of labelling distributions is presented. The method does not require information about compartment size or specimen magnification. It relies on multistage random sampling of specimens and unbiased counting of gold particles associated with different compartments. Distributions of observed gold counts in different experimental groups are compared by contingency table analysis with degrees of freedom for chi-squared (chi(2)) values being determined by the numbers of compartments and experimental groups. Compartmental values of chi(2)which contribute substantially to total chi(2)identify the principal subcellular sites of between-group differences. The method is illustrated using datasets from immunolabelling studies on the localization of GLUT1 glucose transporters in cultured human trophoblast cells exposed to different treatments.

Cell Kinetic and Histomorphometric Analysis of Microgravitational Osteopenia: PARE.03B

NASA Technical Reports Server (NTRS)

Roberts, W. Eugene; Garetto, Lawrence P.

1998-01-01

Previous methods of identifying cells undergoing DNA synthesis (S-phase) utilized H-3 thymidine (3HT) autoradiography. 5-Bromo-2'-deoxyuridine (BrdU) immunohistochemistry is a nonradioactive alternative method. This experiment compared the two methods using the nuclear volume model for osteoblast histogenesis in two different embedding media. Twenty Sprague-Dawley rats were used, with half receiving 3HT (1 micro Ci/g) and the other half BrdU (50 microgram/g). Condyies were embedded (one side in paraffin, the other in plastic) and S-phase nuclei were identified using either autoradiography or immunohistochemistry. The fractional distribution of preosteoblast cell types and the percentage of labeled cells (within each cell fraction and label index) were calculated and expressed as mean q standard error. Chi-Square analysis showed only a minor difference in the fractional distribution of cell types. However, there were significant differences (p less than 0.05) by ANOVA, in the nuclear labeling of specific cell types. With the exception of the less-differentiated A+A'cells, more BrdU label was consistently detected in paraffin than in plastic-embedded sections. In general, more nuclei were labeled with 3H-thymidine than with BrdU in both types of embedding media. Labeling index data (labeled cells/total cells sampled x 100) indicated that BrdU in paraffin, but not plastic gave the same results as 3HT in either embedding method. Thus, we conclude that the two labeling methods do not yield the same results for the nuclear volume model and that embedding media is an important factor whenusing BrdU. As a result of this work, 3HT was chosen for used in the PARE.03 flight experiments.

Comparison of doubly labeled water with respirometry at low- and high-activity levels

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

Westerterp, K.R.; Brouns, F.; Saris, W.H.

1988-07-01

In previous studies the doubly labeled water method for measuring energy expenditure in free-living humans has been validated against respirometry under sedentary conditions. In the present investigation, energy expenditure is measured simultaneously with doubly labeled water and respirometry at low- and high-activity levels. Over 6 days, five subjects were measured doing mainly sedentary activities like desk work; their average daily metabolic rate was 1.40 +/- 0.09 (SD) times sleeping metabolic rate. Four subjects were measured twice over 3.5 days, including 2 days with heavy bicycle ergometer work, resulting in an average daily metabolic rate of 2.61 +/- 0.25 (SD) timesmore » sleeping metabolic rate. At the low-activity level, energy expenditures from the doubly labeled water method were on the average 1.4 +/- 3.9% (SD) larger than those from respirometry. At the high-activity level, the doubly labeled water method yielded values that were 1.0 +/- 7.0% (SD) lower than those from respirometry. Results demonstrate the utility of the doubly labeled water method for the determination of energy expenditure in the range of activity levels in daily life.« less

Stable isotope labelling methods in mass spectrometry-based quantitative proteomics.

PubMed

Chahrour, Osama; Cobice, Diego; Malone, John

2015-09-10

Mass-spectrometry based proteomics has evolved as a promising technology over the last decade and is undergoing a dramatic development in a number of different areas, such as; mass spectrometric instrumentation, peptide identification algorithms and bioinformatic computational data analysis. The improved methodology allows quantitative measurement of relative or absolute protein amounts, which is essential for gaining insights into their functions and dynamics in biological systems. Several different strategies involving stable isotopes label (ICAT, ICPL, IDBEST, iTRAQ, TMT, IPTL, SILAC), label-free statistical assessment approaches (MRM, SWATH) and absolute quantification methods (AQUA) are possible, each having specific strengths and weaknesses. Inductively coupled plasma mass spectrometry (ICP-MS), which is still widely recognised as elemental detector, has recently emerged as a complementary technique to the previous methods. The new application area for ICP-MS is targeting the fast growing field of proteomics related research, allowing absolute protein quantification using suitable elemental based tags. This document describes the different stable isotope labelling methods which incorporate metabolic labelling in live cells, ICP-MS based detection and post-harvest chemical label tagging for protein quantification, in addition to summarising their pros and cons. Copyright © 2015 Elsevier B.V. All rights reserved.

Collagenolytic Activity of Dental Plaque Associated with Periodontal Pathology

PubMed Central

Loesche, W. J.; Paunio, K. U.; Woolfolk, M. P.; Hockett, R. N.

1974-01-01

Certain dental plaques, removed from sites of gingival and periodontal pathology in mentally retarded, institutionalized individuals, when incubated in phosphate buffer with Achilles tendon collagen, gave rise to an increase in ninhydrin-positive material. These plaques, while showing great variability, released significantly more ninhydrin-positive material per milligram of plaque (wet weight) than did either the endogenous or heat-treated controls. Certain plaques could also break down soluble, tritiated, labeled collagen isolated from the calvaria of chicken embryos. Bacteroides melaninogenicus and Clostridia histolyticum were found in plaques by either fluorescent antibody or cultural methods. C. histolyticum, when detected, accounted for about 0.01 to 0.1% of the bacteria in plaque. A conspicuous isolate from some plaques was a Bacillus species which rapidly liquefied gelatin. Cell-free supernatants of this organism were able to degrade about 50 to 70% of the soluble collagen when incubated at 36 C. C. histolyticum ATCC 8034 caused an 80% degradation of the collagen under the same conditions of incubation. The Bacillus strains were facultative, could ferment glucose, reduced nitrate to nitrite, and were catalase, indole, and urease negative. The limited taxonomic information for the isolates is compatible with the description given for Bacillus cereus. PMID:4361294

Video-rate hyperspectral two-photon fluorescence microscopy for in vivo imaging

NASA Astrophysics Data System (ADS)

Deng, Fengyuan; Ding, Changqin; Martin, Jerald C.; Scarborough, Nicole M.; Song, Zhengtian; Eakins, Gregory S.; Simpson, Garth J.

2018-02-01

Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam's position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.

A simple dilute and shoot methodology for the identification and quantification of illegal insulin.

PubMed

Vanhee, Celine; Janvier, Steven; Moens, Goedele; Deconinck, Eric; Courselle, Patricia

2016-10-01

The occurrence of illegal medicines is a well-established global problem and concerns mostly small molecules. However, due to the advances in genomics and recombinant expression technologies there is an increased development of polypeptide therapeutics. Insulin is one of the best known polypeptide drug, and illegal versions of this medicine led to lethal incidents in the past. Therefore, it is crucial for the public health sector to develop reliable, efficient, cheap, unbiased and easily applicable active pharmaceutical ingredient (API) identification and quantification strategies for routine analysis of suspected illegal insulins. Here we demonstrate that our combined label-free full scan approach is not only able to distinguish between all those different versions of insulin and the insulins originating from different species, but also able to chromatographically separate human insulin and insulin lispro in conditions that are compatible with mass spectrometry (MS). Additionally, we were also able to selectively quantify the different insulins, including human insulin and insulin lispro according to the validation criteria, put forward by the United Nations (UN), for the analysis of seized illicit drugs. The proposed identification and quantification method is currently being used in our official medicines control laboratory to analyze insulins retrieved from the illegal market.

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

PubMed Central

Peckys, Diana B.; Veith, Gabriel M.; Joy, David C.; de Jonge, Niels

2009-01-01

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory. PMID:20020038

ISBT 128 Standard for Coding Medical Products of Human Origin

PubMed Central

Ashford, Paul; Delgado, Matthew

2017-01-01

Background ISBT 128 is an international standard for the terminology, coding, labeling, and identification of medical products of human origin (MPHO). Full implementation of ISBT 128 improves traceability, transparency, vigilance and surveillance, and interoperability. Methods ICCBBA maintains the ISBT 128 standard through the activities of a network of expert volunteers, including representatives from professional scientific societies, governments and users, to standardize and maintain MPHO identification. These individuals are organized into Technical Advisory Groups and work within a structured framework as part of a quality-controlled standards development process. Results The extensive involvement of international scientific and professional societies in the development of the standard has ensured that ISBT 128 has gained widespread recognition. The user community has developed confidence in the ability of the standard to adapt to new developments in their fields of interest. The standard is fully compatible with Single European Code requirements for tissues and cells and is utilized by many European tissue establishments. ISBT 128's flexibility and robustness has allowed for expansions into subject areas such as cellular therapy, regenerative medicine, and tissue banking. Conclusion ISBT 128 is the internationally recognized standard for coding MPHO and has gained widespread use globally throughout the past two decades. PMID:29344013

Protein addressing on patterned microchip by coupling chitosan electrodeposition and 'electro-click' chemistry.

PubMed

Shi, Xiao-Wen; Qiu, Ling; Nie, Zhen; Xiao, Ling; Payne, Gregory F; Du, Yumin

2013-12-01

Many applications in proteomics and lab-on-chip analysis require methods that guide proteins to assemble at surfaces with high spatial and temporal control. Electrical inputs are particularly convenient to control, and there has been considerable effort to discover simple and generic mechanisms that allow electrical inputs to trigger protein assembly on-demand. Here, we report the electroaddressing of a protein to a patterned surface by coupling two generic electroaddressing mechanisms. First, we electrodeposit the stimuli-responsive film-forming aminopolysaccharide chitosan to form a hydrogel matrix at the electrode surface. After deposition, the matrix is chemically functionalized with alkyne groups. Second, we ''electro-click' an azide-tagged protein to the functionalized matrix using electrical signals to trigger conjugation by Huisgen 1,3-dipolar cycloadditions. Specifically, a cathodic potential is applied to the matrix-coated electrode to reduce Cu(II) to Cu(I) which is required for the click reaction. Using fluorescently-labeled bovine serum albumin as our model, we demonstrate that protein conjugation can be controlled spatially and temporally. We anticipate that the coupling of polysaccharide electrodeposition and electro-click chemistry will provide a simple and generic approach to electroaddress proteins within compatible hydrogel matrices.

Methods for combining payload parameter variations with input environment. [calculating design limit loads compatible with probabilistic structural design criteria

NASA Technical Reports Server (NTRS)

Merchant, D. H.

1976-01-01

Methods are presented for calculating design limit loads compatible with probabilistic structural design criteria. The approach is based on the concept that the desired limit load, defined as the largest load occurring in a mission, is a random variable having a specific probability distribution which may be determined from extreme-value theory. The design limit load, defined as a particular of this random limit load, is the value conventionally used in structural design. Methods are presented for determining the limit load probability distributions from both time-domain and frequency-domain dynamic load simulations. Numerical demonstrations of the method are also presented.

A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor.

PubMed

Nilam, Mohamed; Gribbon, Philip; Reinshagen, Jeanette; Cordts, Kathrin; Schwedhelm, Edzard; Nau, Werner M; Hennig, Andreas

2017-08-01

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6]uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>10 7 M -1 ) than to the substrate L-ornithine (340 M -1 ). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters ( k cat = 0.12 s -1 and K M = 24 µM) and inhibition constants of the two ODC inhibitors α-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.

Manual of praying mantis morphology, nomenclature, and practices (Insecta, Mantodea)

PubMed Central

Brannoch, Sydney K.; Wieland, Frank; Rivera, Julio; Klass, Klaus-Dieter; Olivier Béthoux; Svenson, Gavin J.

2017-01-01

Abstract This study provides a comprehensive review of historical morphological nomenclature used for praying mantis (Mantodea) morphology, which includes citations, original use, and assignment of homology. All referenced structures across historical works correspond to a proposed standard term for use in all subsequent works pertaining to praying mantis morphology and systematics. The new standards are presented with a verbal description in a glossary as well as indicated on illustrations and images. In the vast majority of cases, originally used terms were adopted as the new standard. In addition, historical morphological topographical homology conjectures are considered with discussion on modern interpretations. A new standardized formulation to present foreleg femoral and tibial spines is proposed for clarity based on previous works. In addition, descriptions for methods of collection, curation, genital complex dissection, and labeling are provided to aid in the proper preservation and storage of specimens for longevity and ease of study. Due to the lack of consistent linear morphometric measurement practices in the literature, we have proposed a series of measurements for taxonomic and morphological research. These measurements are presented with figures to provide visual aids with homologous landmarks to ensure compatibility and comparability across the Order. Finally, our proposed method of pinning mantises is presented with a photographical example as well as a video tutorial available at http://mantodearesearch.com. PMID:29200926

Separation of high-resolution samples of overlapping latent fingerprints using relaxation labeling

NASA Astrophysics Data System (ADS)

Qian, Kun; Schott, Maik; Schöne, Werner; Hildebrandt, Mario

2012-06-01

The analysis of latent fingerprint patterns generally requires clearly recognizable friction ridge patterns. Currently, overlapping latent fingerprints pose a major problem for traditional crime scene investigation. This is due to the fact that these fingerprints usually have very similar optical properties. Consequently, the distinction of two or more overlapping fingerprints from each other is not trivially possible. While it is possible to employ chemical imaging to separate overlapping fingerprints, the corresponding methods require sophisticated fingerprint acquisition methods and are not compatible with conventional forensic fingerprint data. A separation technique that is purely based on the local orientation of the ridge patterns of overlapping fingerprints is proposed by Chen et al. and quantitatively evaluated using off-the-shelf fingerprint matching software with mostly artificially composed overlapping fingerprint samples, which is motivated by the scarce availability of authentic test samples. The work described in this paper adapts the approach presented by Chen et al. for its application on authentic high resolution fingerprint samples acquired by a contactless measurement device based on a Chromatic White Light (CWL) sensor. An evaluation of the work is also given, with the analysis of all adapted parameters. Additionally, the separability requirement proposed by Chen et al. is also evaluated for practical feasibility. Our results show promising tendencies for the application of this approach on high-resolution data, yet the separability requirement still poses a further challenge.

A method for improved visual landscape compatibility of mobile home park

Treesearch

Daniel R. Jones

1979-01-01

This paper is a description of a research effort directed to improving the visual image of mobile home parks in the landscape. The study is an application of existing methodologies for measuring scenic quality and visual landscape compatibility to an unsolved problem. The paper summarizes two major areas of investigation: regional location factors based on visual...

Novel Methods for Electromagnetic Simulation and Design

DTIC Science & Technology

2016-08-03

The resulting discretized integral equations are compatible with fast multipoleaccelerated solvers and will form the basis for high fidelity...expansion”) which are high-order, efficient and easy to use on arbitrarily triangulated surfaces. The resulting discretized integral equations are...created a user interface compatible with both low and high order discretizations , and implemented the generalized Debye approach of [4]. The

METHODS FOR DETERMINING EXPOSURE TO AND POTENTIAL ECOLOGICAL EFFECTS OF GENE FLOW FROM GENETICALLY MODIFIED CROPS TO COMPATIBLE RELATIVES

EPA Science Inventory

SCIENCE QUESTIONS:

-Does gene flow occur from genetically modified (GM) crop plants to compatible plants?

-How can it be measured?

-Are there ecological consequences of GM crop gene flow to plant communities?



RESEARCH:

The objectives ...

A universal procedure for primer labelling of amplicons.

PubMed Central

Neilan, B A; Wilton, A N; Jacobs, D

1997-01-01

Detection and visualisation of nucleic acids is integral to genome analyses. Exponential amplification procedures have provided the means for the manipulation of nucleic acid sequences, which were otherwise inaccessible. We describe the development and application of a universal method for the labelling of any PCR product using a single end-labelled primer. Amplification was performed in a single reaction with the resulting amplicon labelled to a high specific activity. The method was adapted to a wide range of PCRs and significantly reduced the expense of such analyses. PMID:9207046

SAIL--stereo-array isotope labeling.

PubMed

Kainosho, Masatsune; Güntert, Peter

2009-11-01

Optimal stereospecific and regiospecific labeling of proteins with stable isotopes enhances the nuclear magnetic resonance (NMR) method for the determination of the three-dimensional protein structures in solution. Stereo-array isotope labeling (SAIL) offers sharpened lines, spectral simplification without loss of information and the ability to rapidly collect and automatically evaluate the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as before. This review gives an overview of stable isotope labeling methods for NMR spectroscopy with proteins and provides an in-depth treatment of the SAIL technology.

Developments in label-free microfluidic methods for single-cell analysis and sorting.

PubMed

Carey, Thomas R; Cotner, Kristen L; Li, Brian; Sohn, Lydia L

2018-04-24

Advancements in microfluidic technologies have led to the development of many new tools for both the characterization and sorting of single cells without the need for exogenous labels. Label-free microfluidics reduce the preparation time, reagents needed, and cost of conventional methods based on fluorescent or magnetic labels. Furthermore, these devices enable analysis of cell properties such as mechanical phenotype and dielectric parameters that cannot be characterized with traditional labels. Some of the most promising technologies for current and future development toward label-free, single-cell analysis and sorting include electronic sensors such as Coulter counters and electrical impedance cytometry; deformation analysis using optical traps and deformation cytometry; hydrodynamic sorting such as deterministic lateral displacement, inertial focusing, and microvortex trapping; and acoustic sorting using traveling or standing surface acoustic waves. These label-free microfluidic methods have been used to screen, sort, and analyze cells for a wide range of biomedical and clinical applications, including cell cycle monitoring, rapid complete blood counts, cancer diagnosis, metastatic progression monitoring, HIV and parasite detection, circulating tumor cell isolation, and point-of-care diagnostics. Because of the versatility of label-free methods for characterization and sorting, the low-cost nature of microfluidics, and the rapid prototyping capabilities of modern microfabrication, we expect this class of technology to continue to be an area of high research interest going forward. New developments in this field will contribute to the ongoing paradigm shift in cell analysis and sorting technologies toward label-free microfluidic devices, enabling new capabilities in biomedical research tools as well as clinical diagnostics. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices. © 2018 Wiley Periodicals, Inc.

Quantum dot coating of baculoviral vectors enables visualization of transduced cells and tissues

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

Zhao, Ying; Lo, Seong Loong; Zheng, Yuangang

2013-04-26

Highlights: •The use of quantum dot (QD)-labeled viral vectors for in vivo imaging is not well investigated. •A new method to label enveloped baculovirus with glutathione-capped CdTe QDs is developed. •The labeling enables the identification of transduced, cultured cells based on fluorescence. •The labeling also allows evaluation of viral transduction in a real-time manner in living mice. •The method has the potential to assess viral vector-based gene therapy protocols in future. -- Abstract: Imaging of transduced cells and tissues is valuable in developing gene transfer vectors and evaluating gene therapy efficacy. We report here a simple method to use brightmore » and photostable quantum dots to label baculovirus, an emerging gene therapy vector. The labeling was achieved through the non-covalent interaction of glutathione-capped CdTe quantum dots with the virus envelope, without the use of chemical conjugation. The quantum dot labeling was nondestructive to viral transduction function and enabled the identification of baculoviral vector-transduced, living cells based on red fluorescence. When the labeled baculoviral vectors were injected intravenously or intraventricularly for in vivo delivery of a transgene into mice, quantum dot fluorescence signals allow us monitor whether or not the injected tissues were transduced. More importantly, using a dual-color whole-body imaging technology, we demonstrated that in vivo viral transduction could be evaluated in a real-time manner in living mice. Thus, our method of labeling a read-to-use gene delivery vector with quantum dots could be useful towards the improvement of vector design and will have the potential to assess baculovirus-based gene therapy protocols in future.« less

Systematic review: reliability of compendia methods for off-label oncology indications.

PubMed

Abernethy, Amy P; Raman, Gowri; Balk, Ethan M; Hammond, Julia M; Orlando, Lori A; Wheeler, Jane L; Lau, Joseph; McCrory, Douglas C

2009-03-03

The Centers for Medicare & Medicaid Services limit coverage of cancer drugs for off-label indications to indications listed in specified compendia. To assess whether compendia provide comprehensive, research-based, and timely information for off-label prescribing in oncology. 6 drug compendia, English-language literature searches of MEDLINE and the Cochrane Central Register of Controlled Trials from 2006 and 2008, and American Society of Clinical Oncology annual meeting abstracts from 2004 to 2007. Data Assessment: The compendia's stated methods, literature related to off-label indications of 14 cancer drugs in 2006, updated literature related to 1 off-label indication between 2006 and 2008, and completeness of compendia content and citations were assessed. The compendia's stated methods varied greatly from their actual practices. Compendia cited little of the available evidence, often neither the most recent nor that of highest methodological quality. Compendia differed in evidence cited, terminology, detail, presentation, and referencing. For the 14 off-label indications studied, the compendia differed in the indications included and whether and how they recommended particular agents for particular types of cancer. Update schedules varied, and documentation practices made it difficult to determine whether and when compendia content was updated. For 1 indication, compendia citations did not increase between 2006 and 2008 despite newly published articles. The 2006 analysis was limited to 14 off-label indications; the 2008 update examined 1 indication. Only off-label indications for cancer drugs were included, and results cannot be generalized to noncancer drugs or indications. Oncologists rely on compendia for up-to-date access to evidence and reimbursement information for off-label indications. Current compendia lack transparency, cite little current evidence, and lack systematic methods to review or update evidence.

A novel facile method of labeling octreotide with (18)F-fluorine.

PubMed

Laverman, Peter; McBride, William J; Sharkey, Robert M; Eek, Annemarie; Joosten, Lieke; Oyen, Wim J G; Goldenberg, David M; Boerman, Otto C

2010-03-01

Several methods have been developed to label peptides with (18)F. However, in general these are laborious and require a multistep synthesis. We present a facile method based on the chelation of (18)F-aluminum fluoride (Al(18)F) by 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The method is characterized by the labeling of NOTA-octreotide (NOTA-d-Phe-cyclo[Cys-Phe-d-Trp-Lys-Thr-Cys]-Throl (MH(+) 1305) [IMP466]) with (18)F. Octreotide was conjugated with the NOTA chelate and labeled with (18)F in a 2-step, 1-pot method. The labeling procedure was optimized with regard to the labeling buffer, peptide, and aluminum concentration. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of (18)F-IMP466 was studied in AR42J tumor-bearing mice and compared with that of (68)Ga-labeled IMP466. In addition, small-animal PET/CT images were acquired. IMP466 was labeled with Al(18)F in a single step with 50% yield. The labeled product was purified by high-performance liquid chromatography to remove unbound Al(18)F and unlabeled peptide. The radiolabeling, including purification, was performed in 45 min. The specific activity was 45,000 GBq/mmol, and the peptide was stable in serum for 4 h at 37 degrees C. Labeling was performed at pH 4.1 in sodium citrate, sodium acetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 2-(N-morpholino)ethanesulfonic acid buffer and was optimal in sodium acetate buffer. The apparent 50% inhibitory concentration of the (19)F-labeled IMP466 determined on AR42J cells was 3.6 nM. Biodistribution studies at 2 h after injection showed a high tumor uptake of (18)F-IMP466 (28.3 +/- 5.2 percentage injected dose per gram [%ID/g]; tumor-to-blood ratio, 300 +/- 90), which could be blocked by an excess of unlabeled peptide (8.6 +/- 0.7 %ID/g), indicating that the accumulation in the tumor was receptor-mediated. Biodistribution of (68)Ga-IMP466 was similar to that of (18)F-IMP466. (18)F-IMP466 was stable in vivo, because bone uptake was only 0.4 +/- 0.2 %ID/g, whereas free Al(18)F accumulated rapidly in the bone (36.9 +/- 5.0 %ID/g at 2 h after injection). Small-animal PET/CT scans showed excellent tumor delineation and high preferential accumulation in the tumor. NOTA-octreotide could be labeled rapidly and efficiently with (18)F using a 2-step, 1-pot method. The compound was stable in vivo and showed rapid accretion in somatostatin receptor subtype 2-expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds with (18)F.

Facial and semantic emotional interference: A pilot study on the behavioral and cortical responses to the dual valence association task

PubMed Central

2011-01-01

Background Integration of compatible or incompatible emotional valence and semantic information is an essential aspect of complex social interactions. A modified version of the Implicit Association Test (IAT) called Dual Valence Association Task (DVAT) was designed in order to measure conflict resolution processing from compatibility/incompatibly of semantic and facial valence. The DVAT involves two emotional valence evaluative tasks which elicits two forms of emotional compatible/incompatible associations (facial and semantic). Methods Behavioural measures and Event Related Potentials were recorded while participants performed the DVAT. Results Behavioural data showed a robust effect that distinguished compatible/incompatible tasks. The effects of valence and contextual association (between facial and semantic stimuli) showed early discrimination in N170 of faces. The LPP component was modulated by the compatibility of the DVAT. Conclusions Results suggest that DVAT is a robust paradigm for studying the emotional interference effect in the processing of simultaneous information from semantic and facial stimuli. PMID:21489277

Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

PubMed Central

Nelson, P S; Kent, M; Muthini, S

1992-01-01

Novel CE-phosphoramidite (7a-e) and CPG (8a, c, d, e) reagents have been prepared from a unique 2-aminobutyl-1,3-propanediol backbone. The reagents have been used to directly label oligonucleotides with fluorescein, acridine, and biotin via automated DNA synthesis. The versatile 2-aminobutyl-1,3-propanediol backbone allows for labeling at any position (5', internal, and 3') during solid phase oligonucleotide synthesis. Multiple labels can be achieved by repetitive coupling cycles. Furthermore, the 3-carbon atom internucleotide phosphate distance is retained when inserted internally. Using this method, individual oligonucleotides possessing two and three different reporter molecules have been prepared. PMID:1475185

Proximity-Induced Covalent Labeling of Proteins with a Reactive Fluorophore-Binding Peptide Tag.

PubMed

Sunbul, Murat; Nacheva, Lora; Jäschke, Andres

2015-08-19

Labeling of proteins with fluorescent dyes in live cells enables the investigation of their roles in biological systems by fluorescence microscopy. Because the labeling procedure should not disturb the native function of the protein of interest, it is of high importance to find the optimum labeling method for the problem to be studied. Here, we developed a rapid one-step method to covalently and site-specifically label proteins with a TexasRed fluorophore in vitro and in live bacteria. To this end, a genetically encodable TexasRed fluorophore-binding peptide (TR512) was converted into a reactive tag (ReacTR) by adjoining a cysteine residue which rapidly reacts with N-α-chloroacetamide-conjugated TexasRed fluorophore owing to the proximity effect; ReacTR tag first binds to the TexasRed fluorophore and this interaction brings the nucleophilic cysteine and the electrophilic N-α-chloroacetamide groups in close proximity. Our method has several advantages over existing methods: (i) it utilizes a peptide tag much smaller than fluorescent proteins, the SNAP, CLIP, or HaLo tags; (ii) it allows for labeling of proteins with a small, photostable, red-emitting TexasRed fluorophore; (iii) the probe used is very easy to synthesize; (iv) no enzyme is required to transfer the fluorophore to the peptide tag; and (v) labeling yields a stable covalent product in a very fast reaction.

Gold Nanoparticle Labels Amplify Ellipsometric Signals

NASA Technical Reports Server (NTRS)

Venkatasubbarao, Srivatsa

2008-01-01

The ellipsometric method reported in the immediately preceding article was developed in conjunction with a method of using gold nanoparticles as labels on biomolecules that one seeks to detect. The purpose of the labeling is to exploit the optical properties of the gold nanoparticles in order to amplify the measurable ellipsometric effects and thereby to enable ultrasensitive detection of the labeled biomolecules without need to develop more-complex ellipsometric instrumentation. The colorimetric, polarization, light-scattering, and other optical properties of nanoparticles depend on their sizes and shapes. In the present method, these size-and-shape-dependent properties are used to magnify the polarization of scattered light and the diattenuation and retardance of signals derived from ellipsometry. The size-and-shape-dependent optical properties of the nanoparticles make it possible to interrogate the nanoparticles by use of light of various wavelengths, as appropriate, to optimally detect particles of a specific type at high sensitivity. Hence, by incorporating gold nanoparticles bound to biomolecules as primary or secondary labels, the performance of ellipsometry as a means of detecting the biomolecules can be improved. The use of gold nanoparticles as labels in ellipsometry has been found to afford sensitivity that equals or exceeds the sensitivity achieved by use of fluorescence-based methods. Potential applications for ellipsometric detection of gold nanoparticle-labeled biomolecules include monitoring molecules of interest in biological samples, in-vitro diagnostics, process monitoring, general environmental monitoring, and detection of biohazards.

Post-transcriptional labeling by using Suzuki-Miyaura cross-coupling generates functional RNA probes.

PubMed

Walunj, Manisha B; Tanpure, Arun A; Srivatsan, Seergazhi G

2018-06-20

Pd-catalyzed C-C bond formation, an important vertebra in the spine of synthetic chemistry, is emerging as a valuable chemoselective transformation for post-synthetic functionalization of biomacromolecules. While methods are available for labeling protein and DNA, development of an analogous procedure to label RNA by cross-coupling reactions remains a major challenge. Herein, we describe a new Pd-mediated RNA oligonucleotide (ON) labeling method that involves post-transcriptional functionalization of iodouridine-labeled RNA transcripts by using Suzuki-Miyaura cross-coupling reaction. 5-Iodouridine triphosphate (IUTP) is efficiently incorporated into RNA ONs at one or more sites by T7 RNA polymerase. Further, using a catalytic system made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (ADHP) or dimethylamino-substituted ADHP (DMADHP), we established a modular method to functionalize iodouridine-labeled RNA ONs in the presence of various boronic acid and ester substrates under very mild conditions (37°C and pH 8.5). This method is highly chemoselective, and offers direct access to RNA ONs labeled with commonly used fluorescent and affinity tags and new fluorogenic environment-sensitive nucleoside probes in a ligand-controlled stereoselective fashion. Taken together, this simple approach of generating functional RNA ON probes by Suzuki-Miyaura coupling will be a very important addition to the resources and tools available for analyzing RNA motifs.

Safety and efficacy in parous women of a 52-mg levonorgestrel-medicated intrauterine device: a 7-year randomized comparative study with the TCu380A.

PubMed

Rowe, Patrick; Farley, Tim; Peregoudov, Alexandre; Piaggio, Gilda; Boccard, Simone; Landoulsi, Sihem; Meirik, Olav

2016-06-01

To compare rates of unintended pregnancy, method continuation and reasons for removal among women using the 52-mg levonorgestrel (daily release 20 microg) levonorgestrel IUD (LNG-IUD) or the copper T 380A (TCu380A) intrauterine device. This was an open-label 7-year randomized controlled trial in 20 centres, 11 of which in China. Data on 1884 women with interval insertion of the LNG-IUD and 1871 of the TCu380A were analysed using life tables with 30-day intervals and Cox proportional hazards models. The cumulative 7-year pregnancy rate of the LNG-IUD was 0.5 (standard error 0.2) per 100, significantly lower than 2.5 (0.4) per 100 of the TCu380A, cumulative method discontinuation rates at 7 years were 70.6 (1.2) and 40.8 (1.3) per 100, respectively. Dominant reasons for discontinuing the LNG-IUD were amenorrhea (26.1 [1.3] per 100) and reduced bleeding (12.5 [1.1] per 100), particularly in Chinese women and, for the TCu380A, increased bleeding (9.9 [0.9] per 100), especially among non-Chinese women. Removal rates for pain were similar for the two intrauterine devices (IUDs). Cumulative rates of removal for symptoms compatible with hormonal side effects were 5.7 (0.7) and 0.4 (0.2) per 100 for the LNG-IUD and TCu380A, respectively, and cumulative losses to follow-up at 7 years were 26.0 (1.4) and 36.9 (1.3) per 100, respectively. The LNG-IUD and the TCu380A have very high contraceptive efficacy, with the LNG-IUD significantly higher than the TCu380A. Overall rates of IUD removals were higher among LNG-IUD users than TCu380A users. Removals for amenorrhea appeared culturally associated. The 52-mg LNG-IUD and the TCu380A have very high contraceptive efficacy through 7 years. As an IUD, the unique side effects of the LNG-IUD are reduced bleeding, amenorrhea and symptoms compatible with hormonal contraceptives. Copyright © 2016. Published by Elsevier Inc.

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based β-lactam probes.

PubMed

Sadhu, Kalyan K; Mizukami, Shin; Watanabe, Shuji; Kikuchi, Kazuya

2011-05-01

Development of protein labeling techniques with small molecules is enthralling because this method brings promises for triumph over the limitations of fluorescent proteins in live cell imaging. This technology deals with the functionalization of proteins with small molecules and is anticipated to facilitate the expansion of various protein assay methods. A new straightforward aggregation and elimination-based technique for a protein labeling system has been developed with a versatile emissive range of fluorophores. These fluorophores have been applied to show their efficiency for protein labeling by exploiting the same basic principle. A genetically modified version of class A type β-lactamase has been used as the tag protein (BL-tag). The strength of the aggregation interaction between a fluorophore and a quencher plays a governing role in the elimination step of the quencher from the probes, which ultimately controls the swiftness of the protein labeling strategy. Modulation in the elimination process can be accomplished by the variation in the nature of the fluorophore. This diversity facilitates the study of the competitive binding order among the synthesized probes toward the BL-tag labeling method. An aggregation and elimination-based BL-tag technique has been explored to develop an order of color labeling from the equimolar mixture of the labeling probe in solutions. The qualitative and quantitative determination of ordering within the probes toward labeling studies has been executed through SDS-PAGE and time-dependent fluorescence intensity enhancement measurements, respectively. The desirable multiple-wavelength fluorescence labeling probes for the BL-tag technology have been developed and demonstrate broad applicability of this labeling technology to live cell imaging with coumarin and fluorescein derivatives by using confocal microscopy.

Development of a facile and sensitive HPLC-FLD method via fluorescence labeling for triterpenic acid bioavailability investigation.

PubMed

You, Jinmao; Wu, Di; Zhao, Mei; Li, Guoliang; Gong, Peiwei; Wu, Yueyue; Guo, Yu; Chen, Guang; Zhao, Xianen; Sun, Zhiwei; Xia, Lian; Wu, Yongning

2017-06-01

Triterpenic acids are widely distributed in many fruits and are known for their medicinal benefits. The study of bioavailability has been an important task for a better understanding of the triterpenic acids. Although many methods based on fluorescence labeling for triterpenic acid determination have been established, these reported methods needed anhydrous conditions, which are not suitable for the convenient study of triterpenic acid bioavailability. Inspired by that, a versatile method, which overcomes the difficulty of the reported methods, has been first developed in this study. The novel method using 2-[12-benzo[b]acridin-5- (12H)-yl]-acetohydrazide (BAAH) as the fluorescence labeling reagent coupled with high-performance liquid chromatography with fluorescence detection was first developed for the study of triterpenic acid bioavailability. Furthermore, the labeling conditions have been optimized in order to achieve the best fluorescence labeling yield. Under the optimal conditions, the quantitative linear range of analytes was 2-1000 ng mL -1 , and the correlation coefficients were >0.9998. The detection limits for all triterpenic acid derivatives were achieved within the range of 0.28-0.29 ng mL -1 . The proposed method was successfully applied to the study of triterpenic acid bioavailability with excellent applicability and good reproducibility. Copyright © 2016 John Wiley & Sons, Ltd.

Compatibility of segmented thermoelectric generators

NASA Technical Reports Server (NTRS)

Snyder, J.; Ursell, T.

2002-01-01

It is well known that power generation efficiency improves when materials with appropriate properties are combined either in a cascaded or segmented fashion across a temperature gradient. Past methods for determining materials used in segmentation weremainly concerned with materials that have the highest figure of merit in the temperature range. However, the example of SiGe segmented with Bi2Te3 and/or various skutterudites shows a marked decline in device efficiency even though SiGe has the highest figure of merit in the temperature range. The origin of the incompatibility of SiGe with other thermoelectric materials leads to a general definition of compatibility and intrinsic efficiency. The compatibility factor derived as = (Jl+zr - 1) a is a function of only intrinsic material properties and temperature, which is represented by a ratio of current to conduction heat. For maximum efficiency the compatibility factor should not change with temperature both within a single material, and in the segmented leg as a whole. This leads to a measure of compatibility not only between segments, but also within a segment. General temperature trends show that materials are more self compatible at higher temperatures, and segmentation is more difficult across a larger -T. The compatibility factor can be used as a quantitative guide for deciding whether a material is better suited for segmentation orcascading. Analysis of compatibility factors and intrinsic efficiency for optimal segmentation are discussed, with intent to predict optimal material properties, temperature interfaces, and/or currentheat ratios.

Improving condition severity classification with an efficient active learning based framework.

PubMed

Nissim, Nir; Boland, Mary Regina; Tatonetti, Nicholas P; Elovici, Yuval; Hripcsak, George; Shahar, Yuval; Moskovitch, Robert

2016-06-01

Classification of condition severity can be useful for discriminating among sets of conditions or phenotypes, for example when prioritizing patient care or for other healthcare purposes. Electronic Health Records (EHRs) represent a rich source of labeled information that can be harnessed for severity classification. The labeling of EHRs is expensive and in many cases requires employing professionals with high level of expertise. In this study, we demonstrate the use of Active Learning (AL) techniques to decrease expert labeling efforts. We employ three AL methods and demonstrate their ability to reduce labeling efforts while effectively discriminating condition severity. We incorporate three AL methods into a new framework based on the original CAESAR (Classification Approach for Extracting Severity Automatically from Electronic Health Records) framework to create the Active Learning Enhancement framework (CAESAR-ALE). We applied CAESAR-ALE to a dataset containing 516 conditions of varying severity levels that were manually labeled by seven experts. Our dataset, called the "CAESAR dataset," was created from the medical records of 1.9 million patients treated at Columbia University Medical Center (CUMC). All three AL methods decreased labelers' efforts compared to the learning methods applied by the original CAESER framework in which the classifier was trained on the entire set of conditions; depending on the AL strategy used in the current study, the reduction ranged from 48% to 64% that can result in significant savings, both in time and money. As for the PPV (precision) measure, CAESAR-ALE achieved more than 13% absolute improvement in the predictive capabilities of the framework when classifying conditions as severe. These results demonstrate the potential of AL methods to decrease the labeling efforts of medical experts, while increasing accuracy given the same (or even a smaller) number of acquired conditions. We also demonstrated that the methods included in the CAESAR-ALE framework (Exploitation and Combination_XA) are more robust to the use of human labelers with different levels of professional expertise. Copyright © 2016 Elsevier Inc. All rights reserved.

Hierarchical clustering method for improved prostate cancer imaging in diffuse optical tomography

NASA Astrophysics Data System (ADS)

Kavuri, Venkaiah C.; Liu, Hanli

2013-03-01

We investigate the feasibility of trans-rectal near infrared (NIR) based diffuse optical tomography (DOT) for early detection of prostate cancer using a transrectal ultrasound (TRUS) compatible imaging probe. For this purpose, we designed a TRUS-compatible, NIR-based image system (780nm), in which the photo diodes were placed on the trans-rectal probe. DC signals were recorded and used for estimating the absorption coefficient. We validated the system using laboratory phantoms. For further improvement, we also developed a hierarchical clustering method (HCM) to improve the accuracy of image reconstruction with limited prior information. We demonstrated the method using computer simulations laboratory phantom experiments.

Theoretical Basis for Dynamic Label Propagation in Stationary Metabolic Networks under Step and Periodic Inputs

PubMed Central

Sokol, Serguei; Portais, Jean-Charles

2015-01-01

The dynamics of label propagation in a stationary metabolic network during an isotope labeling experiment can provide highly valuable information on the network topology, metabolic fluxes, and on the size of metabolite pools. However, major issues, both in the experimental set-up and in the accompanying numerical methods currently limit the application of this approach. Here, we propose a method to apply novel types of label inputs, sinusoidal or more generally periodic label inputs, to address both the practical and numerical challenges of dynamic labeling experiments. By considering a simple metabolic system, i.e. a linear, non-reversible pathway of arbitrary length, we develop mathematical descriptions of label propagation for both classical and novel label inputs. Theoretical developments and computer simulations show that the application of rectangular periodic pulses has both numerical and practical advantages over other approaches. We applied the strategy to estimate fluxes in a simulated experiment performed on a complex metabolic network (the central carbon metabolism of Escherichia coli), to further demonstrate its value in conditions which are close to those in real experiments. This study provides a theoretical basis for the rational interpretation of label propagation curves in real experiments, and will help identify the strengths, pitfalls and limitations of such experiments. The cases described here can also be used as test cases for more general numerical methods aimed at identifying network topology, analyzing metabolic fluxes or measuring concentrations of metabolites. PMID:26641860

The legibility of prescription medication labelling in Canada

PubMed Central

Ahrens, Kristina; Krishnamoorthy, Abinaya; Gold, Deborah; Rojas-Fernandez, Carlos H.

2014-01-01

Introduction: The legibility of medication labelling is a concern for all Canadians, because poor or illegible labelling may lead to miscommunication of medication information and poor patient outcomes. There are currently few guidelines and no regulations regarding print standards on medication labels. This study analyzed sample prescription labels from Ontario, Canada, and compared them with print legibility guidelines (both generic and specific to medication labels). Methods: Cluster sampling was used to randomly select a total of 45 pharmacies in the tri-cities of Kitchener, Waterloo and Cambridge. Pharmacies were asked to supply a regular label with a hypothetical prescription. The print characteristics of patient-critical information were compared against the recommendations for prescription labels by pharmaceutical and health organizations and for print accessibility by nongovernmental organizations. Results: More than 90% of labels followed the guidelines for font style, contrast, print colour and nonglossy paper. However, only 44% of the medication instructions met the minimum guideline of 12-point print size, and none of the drug or patient names met this standard. Only 5% of the labels were judged to make the best use of space, and 51% used left alignment. None of the instructions were in sentence case, as is recommended. Discussion: We found discrepancies between guidelines and current labels in print size, justification, spacing and methods of emphasis. Conclusion: Improvements in pharmacy labelling are possible without moving to new technologies or changing the size of labels and would be expected to enhance patient outcomes. PMID:24847371

Rate-Compatible Protograph LDPC Codes

NASA Technical Reports Server (NTRS)

Nguyen, Thuy V. (Inventor); Nosratinia, Aria (Inventor); Divsalar, Dariush (Inventor)

2014-01-01

Digital communication coding methods resulting in rate-compatible low density parity-check (LDPC) codes built from protographs. Described digital coding methods start with a desired code rate and a selection of the numbers of variable nodes and check nodes to be used in the protograph. Constraints are set to satisfy a linear minimum distance growth property for the protograph. All possible edges in the graph are searched for the minimum iterative decoding threshold and the protograph with the lowest iterative decoding threshold is selected. Protographs designed in this manner are used in decode and forward relay channels.

A robust fingerprint matching algorithm based on compatibility of star structures

NASA Astrophysics Data System (ADS)

Cao, Jia; Feng, Jufu

2009-10-01

In fingerprint verification or identification systems, most minutiae-based matching algorithms suffered from the problems of non-linear distortion and missing or faking minutiae. Local structures such as triangle or k-nearest structure are widely used to reduce the impact of non-linear distortion, but are suffered from missing and faking minutiae. In our proposed method, star structure is used to present local structure. A star structure contains various number of minutiae, thus, it is more robust with missing and faking minutiae. Our method consists of four steps: 1) Constructing star structures at minutia level; 2) Computing similarity score for each structure pair, and eliminating impostor matched pairs which have the low scores. As it is generally assumed that there is only linear distortion in local area, the similarity is defined by rotation and shifting. 3) Voting for remained matched pairs according to the compatibility between them, and eliminating impostor matched pairs which gain few votes. The concept of compatibility is first introduced by Yansong Feng [4], the original definition is only based on triangles. We define the compatibility for star structures to adjust to our proposed algorithm. 4) Computing the matching score, based on the number of matched structures and their voting scores. The score also reflects the fact that, it should get higher score if minutiae match in more intensive areas. Experiments evaluated on FVC 2004 show both effectiveness and efficiency of our methods.

Labeling and Magnetic Resonance Imaging of Exosomes Isolated from Adipose Stem Cells.

PubMed

Busato, Alice; Bonafede, Roberta; Bontempi, Pietro; Scambi, Ilaria; Schiaffino, Lorenzo; Benati, Donatella; Malatesta, Manuela; Sbarbati, Andrea; Marzola, Pasquina; Mariotti, Raffaella

2017-06-19

Adipose stem cells (ASC) represent a promising therapeutic approach for neurodegenerative diseases. Most biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence the surrounding cells. Current development of exosome therapies requires efficient and noninvasive methods to localize, monitor, and track the exosomes. Among imaging methods used for this purpose, magnetic resonance imaging (MRI) has advantages: high spatial resolution, rapid in vivo acquisition, and radiation-free operation. To be detectable with MRI, exosomes must be labeled with MR contrast agents, such as ultra-small superparamagnetic iron oxide nanoparticles (USPIO). Here, we set up an innovative approach for exosome labeling that preserves their morphology and physiological characteristics. We show that by labeling ASC with USPIO before extraction of nanovesicles, the isolated exosomes retain nanoparticles and can be visualized by MRI. The current work aims at validating this novel USPIO-based exosome labeling method by monitoring the efficiency of the labeling with MRI both in ASC and in exosomes. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Labeling viral envelope lipids with quantum dots by harnessing the biotinylated lipid-self-inserted cellular membrane.

PubMed

Lv, Cheng; Lin, Yi; Liu, An-An; Hong, Zheng-Yuan; Wen, Li; Zhang, Zhenfeng; Zhang, Zhi-Ling; Wang, Hanzhong; Pang, Dai-Wen

2016-11-01

Highly efficient labeling of viruses with quantum dots (QDs) is the prerequisite for the long-term tracking of virus invasion at the single virus level to reveal mechanisms of virus infection. As one of the structural components of viruses, viral envelope lipids are hard to be labeled with QDs due to the lack of efficient methods to modify viral envelope lipids. Moreover, it is still a challenge to maintain the intactness and infectivity of labeled viruses. Herein, a mild method has been developed to label viral envelope lipids with QDs by harnessing the biotinylated lipid-self-inserted cellular membrane. Biotinylated lipids can spontaneously insert in cellular membranes of host cells during culture and then be naturally assembled on progeny Pseudorabies virus (PrV) via propagation. The biotinylated PrV can be labeled with streptavidin-conjugated QDs, with a labeling efficiency of ∼90%. Such a strategy to label lipids with QDs can retain the intactness and infectivity of labeled viruses to the largest extent, facilitating the study of mechanisms of virus infection at the single virus level. Copyright © 2016 Elsevier Ltd. All rights reserved.

A new method for the labelling of proteins with radioactive arsenic isotopes

NASA Astrophysics Data System (ADS)

Jennewein, M.; Hermanne, A.; Mason, R. P.; Thorpe, P. E.; Rösch, F.

2006-12-01

Radioarsenic labelled radiopharmaceuticals could be a valuable asset to positron emission tomography. In particular, the long half-lives of 72As ( T=26 h) and 74As ( T=17.8 d) allow to investigate slow physiological or metabolical processes, like the enrichment and distribution of monoclonal antibodies (mab) in tumour tissue. In this work, a new method for the labelling of proteins with various radioactive arsenic isotopes was developed. For this purpose, two proteins, namely a chimeric IgG 3 monoclonal antibody, ch3G4, directed against anionic phospholipids, and Rituxan (Rituximab), were labelled as a proof of principle with no-carrier-added radioarsenic isotopes ( 74As and 77As). The developed labelling chemistry gives high yields (>99.9%), is reliable and could easily be transferred to automated labelling systems in a clinical environment. At least for the mab used in this work, this route of radioarsenic labelling does not affect the immunoreactivity of the product. The arsenic label stays stable for up to 72 h at the molecular mass of the monoclonal antibody, which is in particular relevant to follow the pharmacology and pharmacokinetics of the labelled mab for several days.

99M-technetium labeled macroaggregated human serum albumin pharmaceutical

DOEpatents

Winchell, Harry S.; Barak, Morton; Van Fleet, III, Parmer

1977-05-17

A reagent comprising macroaggregated human serum albumin having dispersed therein particles of stannous tin and a method for instantly making a labeled pharmaceutical therefrom, are disclosed. The labeled pharmaceutical is utilized in organ imaging.

A paper-based Colorimetric Indicator Label using Natural Dye for Monitoring Shrimp Spoilage

NASA Astrophysics Data System (ADS)

Listyarini, A.; Sholihah, W.; Imawan, C.

2018-05-01

Shrimp is a type of perishable food. This study developed a simple indicator label using colorimetric method for monitoring shrimp freshness. This indicator label was made from natural dye extract of Ruellia simplex flowers which immobilized on cellulose paper by dip coating method. The indicator labels were used for examining freshness of shrimp. In this experiment, shrimp were stored in sealed bottles that have been labeled using the indicator and stored at 13 °C, 25 °C, and 40 °C for a certain range of time. Color changes of the indicator labels were observed using digital photography after shrimp storage for 0 h, 2 h, 17 h and 24 h. The color changes that occur were quantified and analyzed using the ImageJ program. The color of the indicator label when detecting the fresh shrimp was pink and after the shrimp spoilage began, the color of the label changed to purple and then became yellow when the shrimp is badly spoilage. The color change rates of label indicator increases as the shrimp storage temperature increased. These results indicate that this label indicator can be used as an indicator of the freshness of shrimps and it is not toxic and safe for food.

Preliminary evaluation of a gel tube agglutination major cross-match method in dogs.

PubMed

Villarnovo, Dania; Burton, Shelley A; Horney, Barbara S; MacKenzie, Allan L; Vanderstichel, Raphaël

2016-09-01

A major cross-match gel tube test is available for use in dogs yet has not been clinically evaluated. This study compared cross-match results obtained using the gel tube and the standard tube methods for canine samples. Study 1 included 107 canine sample donor-recipient pairings cross-match tested with the RapidVet-H method gel tube test and compared results with the standard tube method. Additionally, 120 pairings using pooled sera containing anti-canine erythrocyte antibody at various concentrations were tested with leftover blood from a hospital population to assess sensitivity and specificity of the gel tube method in comparison with the standard method. The gel tube method had a good relative specificity of 96.1% in detecting lack of agglutination (compatibility) compared to the standard tube method. Agreement between the 2 methods was moderate. Nine of 107 pairings showed agglutination/incompatibility on either test, too few to allow reliable calculation of relative sensitivity. Fifty percent of the gel tube method results were difficult to interpret due to sample spreading in the reaction and/or negative control tubes. The RapidVet-H method agreed with the standard cross-match method on compatible samples, but detected incompatibility in some sample pairs that were compatible with the standard method. Evaluation using larger numbers of incompatible pairings is needed to assess diagnostic utility. The gel tube method results were difficult to categorize due to sample spreading. Weak agglutination reactions or other factors such as centrifuge model may be responsible. © 2016 American Society for Veterinary Clinical Pathology.

Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose

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

Armstrong, G.D.; Peppler, M.S.

1987-05-01

We developed a method to produce radioiodinated pertussis toxin (PT) which was active in the goose erythrocyte agglutination and CHO cell assay systems. The procedure used fetuin coupled to agarose to prevent inactivation of the toxin during the iodination reaction. Analysis of the labeled PT by affinity chromatography on fetuin-agarose and wheat germ agglutinin-agarose and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that there were minimal amounts of labeled fetuin or other contaminants in the labeled PT preparations. All five of the subunits of the toxin appeared to be labeled by the procedure. The labeling method will facilitate further investigationsmore » into the nature of the interaction and activity of PT in host tissues.« less

Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy

PubMed Central

Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

2017-01-01

This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy. PMID:29160812

Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy.

PubMed

Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

2017-11-21

This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

Dual-Quantum-Dots-Labeled Lateral Flow Strip Rapidly Quantifies Procalcitonin and C-reactive Protein

NASA Astrophysics Data System (ADS)

Qi, XiaoPing; Huang, YunYe; Lin, ZhongShi; Xu, Liang; Yu, Hao

2016-03-01

In the article, a dual-quantum-dots-labeled (dual-QDs-labeled) lateral flow strip (LFS) method was developed for the simultaneous and rapid quantitative detection of procalcitonin (PCT) and C-reactive protein (CRP) in the blood. Two QD-antibody conjugates with different fluorescence emission spectra were produced and sprayed on the LFS to capture PCT and CRP in the blood. Furthermore, a double antibody sandwich method for PCT and, meanwhile, a competitive inhibition method for CRP were employed in the LFS. For PCT and CRP in serum assayed by the dual-QDs-labeled LFS, their detection sensitivities reached 0.1 and 1 ng/mL, respectively, and their linear quantitative detection ranges were from 0.3 to 200 ng/mL and from 50 to 250 μg/mL, respectively. There was little evidence that the PCT and CRP assays would be interfered with each other. The correlations for testing CRP and PCT in clinical samples were 99.75 and 97.02 %, respectively, between the dual-QDs-labeled LFS we developed and commercial methods. The rapid quantification of PCT and CRP on dual-QDs-labeled LFS is of great clinical value to distinguish inflammation, bacterial infection, or viral infection and to provide guidance for the use of antibiotics or other medicines.

IRMS detection of testosterone manipulated with 13C labeled standards in human urine by removing the labeled 13C.

PubMed

Wang, Jingzhu; Yang, Rui; Yang, Wenning; Liu, Xin; Xing, Yanyi; Xu, Youxuan

2014-12-10

Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ(13)C value). However, (13)C labeled standards can be used to control the δ(13)C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the (13)C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ(13)C values between Andro and ANAD (Δδ(13)CAndro-ANAD, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different (13)C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ(13)CAndro-ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ(13)CAndro-ANAD for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-(13)C labeled standards. Copyright © 2014 Elsevier B.V. All rights reserved.

Crowdsourcing scoring of immunohistochemistry images: Evaluating Performance of the Crowd and an Automated Computational Method

NASA Astrophysics Data System (ADS)

Irshad, Humayun; Oh, Eun-Yeong; Schmolze, Daniel; Quintana, Liza M.; Collins, Laura; Tamimi, Rulla M.; Beck, Andrew H.

2017-02-01

The assessment of protein expression in immunohistochemistry (IHC) images provides important diagnostic, prognostic and predictive information for guiding cancer diagnosis and therapy. Manual scoring of IHC images represents a logistical challenge, as the process is labor intensive and time consuming. Since the last decade, computational methods have been developed to enable the application of quantitative methods for the analysis and interpretation of protein expression in IHC images. These methods have not yet replaced manual scoring for the assessment of IHC in the majority of diagnostic laboratories and in many large-scale research studies. An alternative approach is crowdsourcing the quantification of IHC images to an undefined crowd. The aim of this study is to quantify IHC images for labeling of ER status with two different crowdsourcing approaches, image-labeling and nuclei-labeling, and compare their performance with automated methods. Crowdsourcing- derived scores obtained greater concordance with the pathologist interpretations for both image-labeling and nuclei-labeling tasks (83% and 87%), as compared to the pathologist concordance achieved by the automated method (81%) on 5,338 TMA images from 1,853 breast cancer patients. This analysis shows that crowdsourcing the scoring of protein expression in IHC images is a promising new approach for large scale cancer molecular pathology studies.

Measurement of protein digestibility in humans by a dual-tracer method.

PubMed

Devi, Sarita; Varkey, Aneesia; Sheshshayee, M S; Preston, Thomas; Kurpad, Anura V

2018-06-01

Recent evaluations of the risk of dietary protein deficiency have indicated that protein digestibility may be a key limiting factor in the provision of indispensable amino acids (IAAs), particularly for vulnerable populations living in challenging environments where intestinal dysfunction may exist. Since the digestion of protein occurs only in the small intestine, and the metabolic activity of colonic bacteria confounds measurements at the fecal level, there is a need to develop noninvasive protein digestibility measurements at the ileal level. We used a dual-tracer method with stable isotopes to characterize the digestibility of uniformly labeled [13C]-spirulina protein as a standard protein, in comparison to a mixture of 2H-labeled crystalline amino acids, and then demonstrated the use of this standard protein to measure the digestibility of selected legumes (chick pea and mung bean) through the use of proteins that were intrinsically labeled with 2H. The digestibility of uniformly labeled [13C]-spirulina was first measured in 6 healthy volunteers (3 males and 3 females) by feeding it along with a standard mixture of 2H-labeled amino acids, in a dual-tracer, plateau-fed test meal approach. Next, intrinsically labeled legume protein digestibility was studied with a similar dual-tracer approach, with uniformly labeled [13C]-spirulina as the standard, when processed differently before consumption. The average digestibility of IAA in spirulina protein was 85.2%. The average IAA digestibility of intrinsically 2H-labeled chick pea and mung bean protein was 56.6% and 57.7%, respectively. Dehulling of mung bean before ingestion increased the average IAA digestibility by 9.9% in comparison to whole mung bean digestibility. An innovative, minimally invasive "dual-stable-isotope" method was developed to measure protein digestibility, in which the ingestion of an intrinsically 2H-labeled test protein along with a 13C-labeled standard protein of known digestibility allows for an accurate measure of digestion and absorption of the intrinsically labeled protein. This minimally invasive method is critical to redefining protein quality and will aid in revisiting human protein requirements in different settings and in vulnerable populations. This trial was registered at Clinical Trials Registry-India as CTRI/2017/11/010468.

Expedited Synthesis of Fluorine-18 Labeled Phenols. A Missing Link in PET Radiochemistry

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

Katzenellenbogen, John A.; Zhou, Dong

Fluorine-18 (F-18) is arguably the most valuable radionuclide for positron emission tomographic (PET) imaging. However, while there are many methods for labeling small molecules with F-18 at aliphatic positions and on electron-deficient aromatic rings, there are essentially no reliable and practical methods to label electron-rich aromatic rings such as phenols, with F-18 at high specific activity. This is disappointing because fluorine-labeled phenols are found in many drugs; there are also many interesting plant metabolites and hormones that contain either phenols or other electron-rich aromatic systems such as indoles whose metabolism, transport, and distribution would be interesting to study if theymore » could readily be labeled with F-18. Most approaches to label phenols with F-18 involve the labeling of electron-poor precursor arenes by nucleophilic aromatic substitution, followed by subsequent conversion to phenols by oxidation or other multi-step sequences that are often inefficient and time consuming. Thus, the lack of good methods for labeling phenols and other electron-rich aromatics with F-18 at high specific activity represents a significant methodological gap in F-18 radiochemistry that can be considered a “Missing Link in PET Radiochemistry”. The objective of this research project was to develop and optimize a series of unusual synthetic transformations that will enable phenols (and other electron-rich aromatic systems) to be labeled with F-18 at high specific activity, rapidly, reliably, and conveniently, thereby bridging this gap. Through the studies conducted with support of this project, we have substantially advanced synthetic methodology for the preparation of fluorophenols. Our progress is presented in detail in the sections below, and much has been published or presented publication; other components are being prepared for publication. In essence, we have developed a completely new method to prepare o-fluorophenols from non-aromatic precursors (diazocyclohexenones) by a novel reaction sequence that uses fluoride ion as a precursor and various activating electrophiles, and we have improved methods for the preparation of heterodiaryl iodonium salts. Both methods have been used to prepare interesting potential radiotracers. Other advances have been made in labeling dendrimeric nanoparticle structures of increasing interest for multimodal imaging and in advancing labeling through fluorosilane bonds. Thus, the progress we have made substantially fills the significant gap in PET radiochemistry that we originally identified, and it provides for the field new methodology that can be applied to a number of current challenges, including the preparation of several molecules of interest as radiotracers, such as 2-[18F]Fluoroestradiol (2-FES) and m-fluorotyrosine, which we have illustrated. These methods can be used by any skilled radiochemist interesting in preparing these agents or similar fluorine-18 labeled electron-rich arene systems of interested for PET biological imaging in the most general sense.« less

[Development and Application of Catalytic Tyrosine Modification].

PubMed

Sato, Shinichi; Tsushima, Michihiko; Nakamura, Kosuke; Nakamura, Hiroyuki

2018-01-01

 The chemical labeling of proteins with synthetic probes is a key technique used in chemical biology, protein-based therapy, and material science. Much of the chemical labeling of native proteins, however, depends on the labeling of lysine and cysteine residues. While those methods have significantly contributed to native protein labeling, alternative methods that can modify different amino acid residues are still required. Herein we report the development of a novel methodology of tyrosine labeling, inspired by the luminol chemiluminescence reaction. Tyrosine residues are often exposed on a protein's surface and are thus expected to be good targets for protein functionalization. In our studies so far, we have found that 1) hemin oxidatively activates luminol derivatives as a catalyst, 2) N-methyl luminol derivative specifically forms a covalent bond with a tyrosine residue among the 20 kinds of natural amino acid residues, and 3) the efficiency of tyrosine labeling with N-methyl luminol derivative is markedly improved by using horseradish peroxidase (HRP) as a catalyst. We were able to use molecular oxygen as an oxidant under HRP/NADH conditions. By using these methods, the functionalization of purified proteins was carried out. Because N-methyl luminol derivative is an excellent protein labeling reagent that responds to the activation of peroxidase, this new method is expected to open doors to such biological applications as the signal amplification of HRP-conjugated antibodies and the detection of protein association in combination with peroxidase-tag technology.

Probabilistic atlas based labeling of the cerebral vessel tree

NASA Astrophysics Data System (ADS)

Van de Giessen, Martijn; Janssen, Jasper P.; Brouwer, Patrick A.; Reiber, Johan H. C.; Lelieveldt, Boudewijn P. F.; Dijkstra, Jouke

2015-03-01

Preoperative imaging of the cerebral vessel tree is essential for planning therapy on intracranial stenoses and aneurysms. Usually, a magnetic resonance angiography (MRA) or computed tomography angiography (CTA) is acquired from which the cerebral vessel tree is segmented. Accurate analysis is helped by the labeling of the cerebral vessels, but labeling is non-trivial due to anatomical topological variability and missing branches due to acquisition issues. In recent literature, labeling the cerebral vasculature around the Circle of Willis has mainly been approached as a graph-based problem. The most successful method, however, requires the definition of all possible permutations of missing vessels, which limits application to subsets of the tree and ignores spatial information about the vessel locations. This research aims to perform labeling using probabilistic atlases that model spatial vessel and label likelihoods. A cerebral vessel tree is aligned to a probabilistic atlas and subsequently each vessel is labeled by computing the maximum label likelihood per segment from label-specific atlases. The proposed method was validated on 25 segmented cerebral vessel trees. Labeling accuracies were close to 100% for large vessels, but dropped to 50-60% for small vessels that were only present in less than 50% of the set. With this work we showed that using solely spatial information of the vessel labels, vessel segments from stable vessels (>50% presence) were reliably classified. This spatial information will form the basis for a future labeling strategy with a very loose topological model.

MilQuant: a free, generic software tool for isobaric tagging-based quantitation.

PubMed

Zou, Xiao; Zhao, Minzhi; Shen, Hongyan; Zhao, Xuyang; Tong, Yuanpeng; Wang, Qingsong; Wei, Shicheng; Ji, Jianguo

2012-09-18

Isobaric tagging techniques such as iTRAQ and TMT are widely used in quantitative proteomics and especially useful for samples that demand in vitro labeling. Due to diversity in choices of MS acquisition approaches, identification algorithms, and relative abundance deduction strategies, researchers are faced with a plethora of possibilities when it comes to data analysis. However, the lack of generic and flexible software tool often makes it cumbersome for researchers to perform the analysis entirely as desired. In this paper, we present MilQuant, mzXML-based isobaric labeling quantitator, a pipeline of freely available programs that supports native acquisition files produced by all mass spectrometer types and collection approaches currently used in isobaric tagging based MS data collection. Moreover, aside from effective normalization and abundance ratio deduction algorithms, MilQuant exports various intermediate results along each step of the pipeline, making it easy for researchers to customize the analysis. The functionality of MilQuant was demonstrated by four distinct datasets from different laboratories. The compatibility and extendibility of MilQuant makes it a generic and flexible tool that can serve as a full solution to data analysis of isobaric tagging-based quantitation. Copyright © 2012 Elsevier B.V. All rights reserved.

An innovative large scale integration of silicon nanowire-based field effect transistors

NASA Astrophysics Data System (ADS)

Legallais, M.; Nguyen, T. T. T.; Mouis, M.; Salem, B.; Robin, E.; Chenevier, P.; Ternon, C.

2018-05-01

Since the early 2000s, silicon nanowire field effect transistors are emerging as ultrasensitive biosensors while offering label-free, portable and rapid detection. Nevertheless, their large scale production remains an ongoing challenge due to time consuming, complex and costly technology. In order to bypass these issues, we report here on the first integration of silicon nanowire networks, called nanonet, into long channel field effect transistors using standard microelectronic process. A special attention is paid to the silicidation of the contacts which involved a large number of SiNWs. The electrical characteristics of these FETs constituted by randomly oriented silicon nanowires are also studied. Compatible integration on the back-end of CMOS readout and promising electrical performances open new opportunities for sensing applications.

Chemoselective ligation

DOEpatents

Saxon, Eliana [Albany, CA; Bertozzi, Carolyn Ruth [Berkeley, CA

2011-12-13

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana; Bertozzi, Carolyn

2006-10-17

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana [Albany, CA; Bertozzi, Carolyn R [Berkeley, CA

2011-05-10

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Sorting drops and cells with acoustics: acoustic microfluidic fluorescence-activated cell sorter.

PubMed

Schmid, Lothar; Weitz, David A; Franke, Thomas

2014-10-07

We describe a versatile microfluidic fluorescence-activated cell sorter that uses acoustic actuation to sort cells or drops at ultra-high rates. Our acoustic sorter combines the advantages of traditional fluorescence-activated cell (FACS) and droplet sorting (FADS) and is applicable for a multitude of objects. We sort aqueous droplets, at rates as high as several kHz, into two or even more outlet channels. We can also sort cells directly from the medium without prior encapsulation into drops; we demonstrate this by sorting fluorescently labeled mouse melanoma cells in a single phase fluid. Our acoustic microfluidic FACS is compatible with standard cell sorting cytometers, yet, at the same time, enables a rich variety of more sophisticated applications.

Chemoselective ligation

DOEpatents

Saxon, Eliana; Bertozzi, Carolyn Ruth

2010-11-23

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana [Albany, CA; Bertozzi, Carolyn R [Berkeley, CA

2011-04-12

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana; Bertozzi, Carolyn R.

2010-02-23

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g. on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana [Albany, CA; Bertozzi, Carolyn [Berkeley, CA

2003-05-27

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

[Possibility and necessity of constructing new nanoformula systems of traditional Chinese medicine].

PubMed

Ling, Chang-quan; Su, Yong-hua

2010-02-01

The past decade has witnessed the remarkable progress on nanotechnology and nanoherb. With the globally rapid development of nanotechnology, we are considering to construct new nanoformula systems of traditional Chinese medicine (TCM) by using porous materials, multilayered core-shell particles or nanoparticles containing various multifunctional parts. With the compatibility of sovereign, minister, assistant and courier in a formula, new nanoformula systems of TCM will have various advantages, such as containing multiple active species, controlled release, targeting function, and labeling and tracing capabilities. Using the latest breakthroughs of nanotechnology for the modern research of TCM will greatly help enhance the ability to investigate the principles of TCM, and to design, screen and utilize new nanoformula systems of TCM.

Validation of laboratory-scale recycling test method of paper PSA label products

Treesearch

Carl Houtman; Karen Scallon; Richard Oldack

2008-01-01

Starting with test methods and a specification developed by the U.S. Postal Service (USPS) Environmentally Benign Pressure Sensitive Adhesive Postage Stamp Program, a laboratory-scale test method and a specification were developed and validated for pressure-sensitive adhesive labels, By comparing results from this new test method and pilot-scale tests, which have been...

Evaluation of a High Intensity Focused Ultrasound-Immobilized Trypsin Digestion and 18O-Labeling Method for Quantitative Proteomics

PubMed Central

López-Ferrer, Daniel; Hixson, Kim K.; Smallwood, Heather; Squier, Thomas C.; Petritis, Konstantinos; Smith, Richard D.

2009-01-01

A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultra-rapid digestion and thorough 18O labeling for quantitative protein comparisons. The reproducible and highly efficient method provided effective digestions in <1 min with a minimized amount of enzyme required compared to traditional methods. This method was demonstrated for digestion of both simple and complex protein mixtures, including bovine serum albumin, a global proteome extract from the bacteria Shewanella oneidensis, and mouse plasma, as well as 18O labeling of such complex protein mixtures, which validated the application of this method for differential proteomic measurements. This approach is simple, reproducible, cost effective, rapid, and thus well-suited for automation. PMID:19555078

Survival Time of Cross-Match Incompatible Red Blood Cells in Adult Horses.

PubMed

Tomlinson, J E; Taberner, E; Boston, R C; Owens, S D; Nolen-Walston, R D

2015-01-01

There is a markedly reduced half-life of transfused RBCs when donor and recipient cats or humans are cross-match incompatible. Only 10-20% of horses have naturally occurring alloantibodies. Therefore, cross-match testing before blood transfusion is not always performed. Cross-match incompatibility predicts shortened RBC survival time as compared to that of compatible or autologous blood. Twenty healthy adult horses. Prospective trial. Blood type, anti-RBC antibody screen (before and 1 month after transfusion) and major and minor cross-match determined 10 donor-recipient pairs. Two pairs were cross-match compatible, the remainder incompatible. Donor blood (4 L) was collected into citrate phosphate dextrose adenine-1, labeled with NHS-biotin, and transfused into recipients. Samples were collected at 1 hour and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after transfusion, and biotinylated RBCs were detected by flow cytometry. Horses were monitored for transfusion reaction during transfusion and daily for 5 days. Cross-match incompatibility was significantly associated with decreased RBC survival time (P < .001). The half-life of transfused incompatible (cross-match >1+) allogenic equine RBCs was 4.7 (95% CI, 3.2-6.2) days versus 33.5 (24-43) days for compatible pairings. Cross-match incompatibility was associated with acute febrile transfusion reaction (P = .0083). At day 30, only 1 horse had developed novel anti-RBC antibodies. Cross-match incompatibility was predictive of febrile transfusion reaction and shortened transfused RBC survival, but did not result in production of anti-RBC antibodies at 30 days. Cross-match testing before transfusion is recommended. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Human TSCM cell dynamics in vivo are compatible with long-lived immunological memory and stemness.

PubMed

Del Amo, Pedro Costa; Beneytez, Julio Lahoz; Boelen, Lies; Ahmed, Raya; Miners, Kelly L; Zhang, Yan; Roger, Laureline; Jones, Rhiannon E; Marraco, Silvia A Fuertes; Speiser, Daniel E; Baird, Duncan M; Price, David A; Ladell, Kristin; Macallan, Derek; Asquith, Becca

2018-06-22

Adaptive immunity relies on the generation and maintenance of memory T cells to provide protection against repeated antigen exposure. It has been hypothesised that a self-renewing population of T cells, named stem cell-like memory T (TSCM) cells, are responsible for maintaining memory. However, it is not clear if the dynamics of TSCM cells in vivo are compatible with this hypothesis. To address this issue, we investigated the dynamics of TSCM cells under physiological conditions in humans in vivo using a multidisciplinary approach that combines mathematical modelling, stable isotope labelling, telomere length analysis, and cross-sectional data from vaccine recipients. We show that, unexpectedly, the average longevity of a TSCM clone is very short (half-life < 1 year, degree of self-renewal = 430 days): far too short to constitute a stem cell population. However, we also find that the TSCM population is comprised of at least 2 kinetically distinct subpopulations that turn over at different rates. Whilst one subpopulation is rapidly replaced (half-life = 5 months) and explains the rapid average turnover of the bulk TSCM population, the half-life of the other TSCM subpopulation is approximately 9 years, consistent with the longevity of the recall response. We also show that this latter population exhibited a high degree of self-renewal, with a cell residing without dying or differentiating for 15% of our lifetime. Finally, although small, the population was not subject to excessive stochasticity. We conclude that the majority of TSCM cells are not stem cell-like but that there is a subpopulation of TSCM cells whose dynamics are compatible with their putative role in the maintenance of T cell memory.

[Preparation of acellular matrix from antler cartilage and its biological compatibility].

PubMed

Fu, Jing; Zhang, Wei; Zhang, Aiwu; Ma, Lijuan; Chu, Wenhui; Li, Chunyi

2017-06-01

To study the feasibility of acellular matrix materials prepared from deer antler cartilage and its biological compatibility so as to search for a new member of the extracellular matrix family for cartilage regeneration. The deer antler mesenchymal (M) layer tissue was harvested and treated through decellular process to prepare M layer acellular matrix; histologic observation and detection of M layer acellular matrix DNA content were carried out. The antler stem cells [antlerogenic periosteum (AP) cells] at 2nd passage were labelled by fluorescent stains and by PKH26. Subsequently, the M layer acellular matrix and the AP cells at 2nd passage were co-cultured for 7 days; then the samples were transplanted into nude mice to study the tissue compatibility of M layer acellular matrix in the living animals. HE and DAPI staining confirmed that the M layer acellular matrix did not contain nucleus; the DNA content of the M layer acellular matrix was (19.367±5.254) ng/mg, which was significantly lower than that of the normal M layer tissue [(3 805.500±519.119) ng/mg]( t =12.630, P =0.000). In vitro co-culture experiments showed that AP cells could adhere to or even embedded in the M layer acellular matrix. Nude mice transplantation experiments showed that the introduced AP cells could proliferate and induce angiogenesis in the M layer acellular matrix. The deer antler cartilage acellular matrix is successfully prepared. The M layer acellular matrix is suitable for adhesion and proliferation of AP cells in vitro and in vivo , and it has the function of stimulating angiogenesis. This model for deer antler cartilage acellular matrix can be applied in cartilage tissue engineering in the future.

Preparation of anionic polyurethane nanoparticles and blood compatible behaviors.

PubMed

Zhu, Qinshu; Wang, Yan; Zhou, Min; Mao, Chun; Huang, Xiaohua; Bao, Jianchun; Shen, Jian

2012-05-01

The anionic polyurethane nanoparticles (APU-NPs) were obtained by an emulsion polymerization method. It was found that the average size of the prepared APU-NPs is about 84 nm, and the APU-NPs have zeta-potential of -38.9 mV. The bulk characterization of synthesized APU-NPs was investigated by FTIR. The blood compatibility of APU-NPs was characterized by in vitro for coagulation tests, complement activation, platelet activation, cytotoxicity experiments, and hemolysis assay. The results showed that the APU-NPs synthesized in this paper are blood compatible with low level of cell cytotoxicity, and the results were significant for their potential use in vivo.

Standard addition with internal standardisation as an alternative to using stable isotope labelled internal standards to correct for matrix effects-Comparison and validation using liquid chromatography-​tandem mass spectrometric assay of vitamin D.

PubMed

Hewavitharana, Amitha K; Abu Kassim, Nur Sofiah; Shaw, Paul Nicholas

2018-06-08

With mass spectrometric detection in liquid chromatography, co-eluting impurities affect the analyte response due to ion suppression/enhancement. Internal standard calibration method, using co-eluting stable isotope labelled analogue of each analyte as the internal standard, is the most appropriate technique available to correct for these matrix effects. However, this technique is not without drawbacks, proved to be expensive because separate internal standard for each analyte is required, and the labelled compounds are expensive or require synthesising. Traditionally, standard addition method has been used to overcome the matrix effects in atomic spectroscopy and was a well-established method. This paper proposes the same for mass spectrometric detection, and demonstrates that the results are comparable to those with the internal standard method using labelled analogues, for vitamin D assay. As conventional standard addition procedure does not address procedural errors, we propose the inclusion of an additional internal standard (not co-eluting). Recoveries determined on human serum samples show that the proposed method of standard addition yields more accurate results than the internal standardisation using stable isotope labelled analogues. The precision of the proposed method of standard addition is superior to the conventional standard addition method. Copyright © 2018 Elsevier B.V. All rights reserved.

Single step signal group-imidazole labeling of organic phosphate groups under aqueous conditions

DOEpatents

Giese, Roger W.; Wang, Poguang

1996-01-01

Compounds and methods for single step, covalent labeling of the phosphate group of an organic substance under aqueous conditions are described. The labeling compound includes any kind of detectable signal group covalently bound to an imidazole moiety, which can be imidazole or a substituted imidazole. A preferred labeling compound has the formula ##STR1##

Labeling and Knowing: A Reconciliation of Implicit Theory and Explicit Theory among Students with Exceptionalities

ERIC Educational Resources Information Center

lo, C. Owen

2014-01-01

Using a realist grounded theory method, this study resulted in a theoretical model and 4 propositions. As displayed in the LINK model, the labeling practice is situated in and endorsed by a social context that carries explicit theory about and educational policies regarding the labels. Taking a developmental perspective, the labeling practice…

Potential Impact of ADHD with Stimulant Medication Label on Teacher Expectations

ERIC Educational Resources Information Center

Batzle, Christina S.; Weyandt, Lisa L.; Janusis, Grace M.; DeVietti, Terry L.

2010-01-01

Objective: The present study investigated how teachers rated children's Behavior, IQ, and Personality contingent on the presence or absence of an Attention Deficit Hyperactivity Disorder (ADHD) label. Method: Teachers from K-12 read a hypothetical description of either a male or female child with no label, an ADHD label, or an ADHD with stimulant…

Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants.

PubMed

Weiss, Agnes; Jérôme, Valérie; Freitag, Ruth

2007-06-15

The goal of the project was the extraction of PCR-compatible genomic DNA representative of the entire microbial community from municipal biogas plant samples (mash, bioreactor content, process water, liquid fertilizer). For the initial isolation of representative DNA from the respective lysates, methods were used that employed adsorption, extraction, or precipitation to specifically enrich the DNA. Since no dedicated method for biogas plant samples was available, preference was given to kits/methods suited to samples that resembled either the bioreactor feed, e.g. foodstuffs, or those intended for environmental samples including wastewater. None of the methods succeeded in preparing DNA that was directly PCR-compatible. Instead the DNA was found to still contain considerable amounts of difficult-to-remove enzyme inhibitors (presumably humic acids) that hindered the PCR reaction. Based on the isolation method that gave the highest yield/purity for all sample types, subsequent purification was attempted by agarose gel electrophoresis followed by electroelution, spermine precipitation, or dialysis through nitrocellulose membrane. A combination of phenol/chloroform extraction followed by purification via dialysis constituted the most efficient sample treatment. When such DNA preparations were diluted 1:100 they did no longer inhibit PCR reactions, while they still contained sufficient genomic DNA to allow specific amplification of specific target sequences.

Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection.

PubMed

Chung, Heaseung Sophia; Murray, Christopher I; Venkatraman, Vidya; Crowgey, Erin L; Rainer, Peter P; Cole, Robert N; Bomgarden, Ryan D; Rogers, John C; Balkan, Wayne; Hare, Joshua M; Kass, David A; Van Eyk, Jennifer E

2015-10-23

S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations. © 2015 American Heart Association, Inc.

A Low-Storage-Consumption XML Labeling Method for Efficient Structural Information Extraction

NASA Astrophysics Data System (ADS)

Liang, Wenxin; Takahashi, Akihiro; Yokota, Haruo

Recently, labeling methods to extract and reconstruct the structural information of XML data, which are important for many applications such as XPath query and keyword search, are becoming more attractive. To achieve efficient structural information extraction, in this paper we propose C-DO-VLEI code, a novel update-friendly bit-vector encoding scheme, based on register-length bit operations combining with the properties of Dewey Order numbers, which cannot be implemented in other relevant existing schemes such as ORDPATH. Meanwhile, the proposed method also achieves lower storage consumption because it does not require either prefix schema or any reserved codes for node insertion. We performed experiments to evaluate and compare the performance and storage consumption of the proposed method with those of the ORDPATH method. Experimental results show that the execution times for extracting depth information and parent node labels using the C-DO-VLEI code are about 25% and 15% less, respectively, and the average label size using the C-DO-VLEI code is about 24% smaller, comparing with ORDPATH.

A cost-effective approach to produce 15N-labelled amino acids employing Chlamydomonas reinhardtii CC503.

PubMed

Nicolás Carcelén, Jesús; Marchante-Gayón, Juan Manuel; González, Pablo Rodríguez; Valledor, Luis; Cañal, María Jesús; Alonso, José Ignacio García

2017-08-18

The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15 N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15 NH 4 Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15 N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13 C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen 15 N-labelled amino acids could be obtained.

High efficiency labeling of glycoproteins on living cells

PubMed Central

Zeng, Ying; Ramya, T. N. C.; Dirksen, Anouk; Dawson, Philip E.; Paulson, James C.

2010-01-01

We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. PMID:19234450

Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

PubMed Central

Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

2014-01-01

Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP). PMID:25154394

Streaming video-based 3D reconstruction method compatible with existing monoscopic and stereoscopic endoscopy systems

NASA Astrophysics Data System (ADS)

Bouma, Henri; van der Mark, Wannes; Eendebak, Pieter T.; Landsmeer, Sander H.; van Eekeren, Adam W. M.; ter Haar, Frank B.; Wieringa, F. Pieter; van Basten, Jean-Paul

2012-06-01

Compared to open surgery, minimal invasive surgery offers reduced trauma and faster recovery. However, lack of direct view limits space perception. Stereo-endoscopy improves depth perception, but is still restricted to the direct endoscopic field-of-view. We describe a novel technology that reconstructs 3D-panoramas from endoscopic video streams providing a much wider cumulative overview. The method is compatible with any endoscope. We demonstrate that it is possible to generate photorealistic 3D-environments from mono- and stereoscopic endoscopy. The resulting 3D-reconstructions can be directly applied in simulators and e-learning. Extended to real-time processing, the method looks promising for telesurgery or other remote vision-guided tasks.

High-Throughput Density Measurement Using Magnetic Levitation.

PubMed

Ge, Shencheng; Wang, Yunzhe; Deshler, Nicolas J; Preston, Daniel J; Whitesides, George M

2018-06-20

This work describes the development of an integrated analytical system that enables high-throughput density measurements of diamagnetic particles (including cells) using magnetic levitation (MagLev), 96-well plates, and a flatbed scanner. MagLev is a simple and useful technique with which to carry out density-based analysis and separation of a broad range of diamagnetic materials with different physical forms (e.g., liquids, solids, gels, pastes, gums, etc.); one major limitation, however, is the capacity to perform high-throughput density measurements. This work addresses this limitation by (i) re-engineering the shape of the magnetic fields so that the MagLev system is compatible with 96-well plates, and (ii) integrating a flatbed scanner (and simple optical components) to carry out imaging of the samples that levitate in the system. The resulting system is compatible with both biological samples (human erythrocytes) and nonbiological samples (simple liquids and solids, such as 3-chlorotoluene, cholesterol crystals, glass beads, copper powder, and polymer beads). The high-throughput capacity of this integrated MagLev system will enable new applications in chemistry (e.g., analysis and separation of materials) and biochemistry (e.g., cellular responses under environmental stresses) in a simple and label-free format on the basis of a universal property of all matter, i.e., density.

CMOS-compatible silicon nanowire field-effect transistors for ultrasensitive and label-free microRNAs sensing.

PubMed

Lu, Na; Gao, Anran; Dai, Pengfei; Song, Shiping; Fan, Chunhai; Wang, Yuelin; Li, Tie

2014-05-28

MicroRNAs (miRNAs) have been regarded as promising biomarkers for the diagnosis and prognosis of early-stage cancer as their expression levels are associated with different types of human cancers. However, it is a challenge to produce low-cost miRNA sensors, as well as retain a high sensitivity, both of which are essential factors that must be considered in fabricating nanoscale biosensors and in future biomedical applications. To address such challenges, we develop a complementary metal oxide semiconductor (CMOS)-compatible SiNW-FET biosensor fabricated by an anisotropic wet etching technology with self-limitation which provides a much lower manufacturing cost and an ultrahigh sensitivity. This nanosensor shows a rapid (< 1 minute) detection of miR-21 and miR-205, with a low limit of detection (LOD) of 1 zeptomole (ca. 600 copies), as well as an excellent discrimination for single-nucleotide mismatched sequences of tumor-associated miRNAs. To investigate its applicability in real settings, we have detected miRNAs in total RNA extracted from lung cancer cells as well as human serum samples using the nanosensors, which demonstrates their potential use in identifying clinical samples for early diagnosis of cancer. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-aligned Multi-task Feature Learning for Multimodal Classification of Alzheimer’s Disease and Mild Cognitive Impairment

PubMed Central

Zu, Chen; Jie, Biao; Liu, Mingxia; Chen, Songcan

2015-01-01

Multimodal classification methods using different modalities of imaging and non-imaging data have recently shown great advantages over traditional single-modality-based ones for diagnosis and prognosis of Alzheimer’s disease (AD), as well as its prodromal stage, i.e., mild cognitive impairment (MCI). However, to the best of our knowledge, most existing methods focus on mining the relationship across multiple modalities of the same subjects, while ignoring the potentially useful relationship across different subjects. Accordingly, in this paper, we propose a novel learning method for multimodal classification of AD/MCI, by fully exploring the relationships across both modalities and subjects. Specifically, our proposed method includes two subsequent components, i.e., label-aligned multi-task feature selection and multimodal classification. In the first step, the feature selection learning from multiple modalities are treated as different learning tasks and a group sparsity regularizer is imposed to jointly select a subset of relevant features. Furthermore, to utilize the discriminative information among labeled subjects, a new label-aligned regularization term is added into the objective function of standard multi-task feature selection, where label-alignment means that all multi-modality subjects with the same class labels should be closer in the new feature-reduced space. In the second step, a multi-kernel support vector machine (SVM) is adopted to fuse the selected features from multi-modality data for final classification. To validate our method, we perform experiments on the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database using baseline MRI and FDG-PET imaging data. The experimental results demonstrate that our proposed method achieves better classification performance compared with several state-of-the-art methods for multimodal classification of AD/MCI. PMID:26572145

Compatibility testing of spacecraft materials and spacestorable liquid propellants. [liquid and vapor fluorine and FLOX

NASA Technical Reports Server (NTRS)

Denson, J. R.; Toy, A.

1974-01-01

Compatibility data for aluminum alloy 2219-T87 and titanium alloy Ti-6Al-4V were obtained while these alloys were exposed to both liquid and vapor fluorine and FLOX at -320 F + or -10 F. These data were obtained using a new low cost compatibility method which incorporates totally sealed containers and double dogbone test specimens and propellants in the simultaneous exposure to vapor and liquid phases. The compatibility investigation covered a storage period in excess of one year. Pitting was more severe in the 2219-T87 aluminum alloy than in the Ti-6Al-4V titanium alloy for both fluorine and FLOX exposure. The degree of chemical attack is more severe in the presence of FLOX than in fluorine and phase. The mechanical properties of the two alloys were not affected by storage in either of the two propellants.

New advances in MR-compatible bioartificial liver

PubMed Central

Jeffries, Rex E.; Macdonald, Jeffrey M.

2015-01-01

MR-compatible bioartificial liver (BAL) studies have been performed for 30 years and are reviewed. There are two types of study: (i) metabolism and drug studies using multinuclear MRS; primarily short-term (< 8 h) studies; (ii) the use of multinuclear MRS and MRI to noninvasively define the features and functions of BAL systems for long-term liver tissue engineering. In the latter, these systems often undergo not only modification of the perfusion system, but also the construction of MR radiofrequency probes around the bioreactor. We present novel MR-compatible BALs and the use of multinuclear MRS (13C, 19F, 31P) for the noninvasive monitoring of their growth, metabolism and viability, as well as 1H MRI methods for the determination of flow profiles, diffusion, cell distribution, quality assurance and bioreactor integrity. Finally, a simple flexible coil design and circuit, and life support system, are described that can make almost any BAL MR-compatible. PMID:22351642

Optimization and validation of FePro cell labeling method.

PubMed

Janic, Branislava; Rad, Ali M; Jordan, Elaine K; Iskander, A S M; Ali, Md M; Varma, N Ravi S; Frank, Joseph A; Arbab, Ali S

2009-06-11

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 microg/ml of Fe and 3 microg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 microg/ml of Fe and 3 microg/ml of Pro is effective in labeling cells for cellular MRI.

Semi-automatic ground truth generation using unsupervised clustering and limited manual labeling: Application to handwritten character recognition

PubMed Central

Vajda, Szilárd; Rangoni, Yves; Cecotti, Hubert

2015-01-01

For training supervised classifiers to recognize different patterns, large data collections with accurate labels are necessary. In this paper, we propose a generic, semi-automatic labeling technique for large handwritten character collections. In order to speed up the creation of a large scale ground truth, the method combines unsupervised clustering and minimal expert knowledge. To exploit the potential discriminant complementarities across features, each character is projected into five different feature spaces. After clustering the images in each feature space, the human expert labels the cluster centers. Each data point inherits the label of its cluster’s center. A majority (or unanimity) vote decides the label of each character image. The amount of human involvement (labeling) is strictly controlled by the number of clusters – produced by the chosen clustering approach. To test the efficiency of the proposed approach, we have compared, and evaluated three state-of-the art clustering methods (k-means, self-organizing maps, and growing neural gas) on the MNIST digit data set, and a Lampung Indonesian character data set, respectively. Considering a k-nn classifier, we show that labeling manually only 1.3% (MNIST), and 3.2% (Lampung) of the training data, provides the same range of performance than a completely labeled data set would. PMID:25870463

Manifold regularized matrix completion for multi-label learning with ADMM.

PubMed

Liu, Bin; Li, Yingming; Xu, Zenglin

2018-05-01

Multi-label learning is a common machine learning problem arising from numerous real-world applications in diverse fields, e.g, natural language processing, bioinformatics, information retrieval and so on. Among various multi-label learning methods, the matrix completion approach has been regarded as a promising approach to transductive multi-label learning. By constructing a joint matrix comprising the feature matrix and the label matrix, the missing labels of test samples are regarded as missing values of the joint matrix. With the low-rank assumption of the constructed joint matrix, the missing labels can be recovered by minimizing its rank. Despite its success, most matrix completion based approaches ignore the smoothness assumption of unlabeled data, i.e., neighboring instances should also share a similar set of labels. Thus they may under exploit the intrinsic structures of data. In addition, the matrix completion problem can be less efficient. To this end, we propose to efficiently solve the multi-label learning problem as an enhanced matrix completion model with manifold regularization, where the graph Laplacian is used to ensure the label smoothness over it. To speed up the convergence of our model, we develop an efficient iterative algorithm, which solves the resulted nuclear norm minimization problem with the alternating direction method of multipliers (ADMM). Experiments on both synthetic and real-world data have shown the promising results of the proposed approach. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development of Design Standards and Guidelines for Electromagnetic Compatibility and Lightning Protection for Spacecraft Utilizing Composite Materials

NASA Technical Reports Server (NTRS)

1996-01-01

Preliminary design guidelines necessary to assure electromagnetic compatibility (EMC) of spacecraft using composite materials, are presented. A database of electrical properties of composite materials which may have an effect on EMC is established. The guidelines concentrate on the composites that are conductive but may require enhancement to be adequate for EMC purposes. These composites are represented by graphite reinforced polymers. Methods for determining adequate conductivity levels for various EMC purposes are defined, along with the methods of design which increase conductivity of composite materials and joints to adequate levels.

The oxygen sensitivity/compatibility ranking of several materials by different test methods

NASA Technical Reports Server (NTRS)

Lockhart, Billy J.; Bryan, Coleman J.; Hampton, Michael D.

1989-01-01

Eleven materials were evaluated for oxygen compatibility using the following test methods: heat of combustion (ASTM D 2015), liquid oxygen impact (ASTM D 2512), pneumatic impact (ASTM G 74), gaseous mechanical impact (ASTM G 86), autogenous ignition temperature by pressurized differential scanning calorimeter, and the determination of the 50 percent reaction level in liquid oxygen using silicon carbide as a reaction enhancer. The eleven materials evaluated were: Teflon TFE, Vespel SP-21, Krytox 240AC, Viton PLV5010B, Fluorel E2160, Kel F 81, Fluorogold, Fluorogreen E-600, Rulon A, Garlock 8573, nylon 6/6.

Reductive methods for isotopic labeling of antibiotics

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

Champney, W.S.

1989-08-15

Methods for the reductive methylation of the amino groups of eight different antibiotics using {sup 3}HCOH or H{sup 14}COH are presented. The reductive labeling of an additional seven antibiotics by NaB{sub 3}H{sub 4} is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented.

Advancing The Cancer Genome Atlas glioma MRI collections with expert segmentation labels and radiomic features

PubMed Central

Bakas, Spyridon; Akbari, Hamed; Sotiras, Aristeidis; Bilello, Michel; Rozycki, Martin; Kirby, Justin S.; Freymann, John B.; Farahani, Keyvan; Davatzikos, Christos

2017-01-01

Gliomas belong to a group of central nervous system tumors, and consist of various sub-regions. Gold standard labeling of these sub-regions in radiographic imaging is essential for both clinical and computational studies, including radiomic and radiogenomic analyses. Towards this end, we release segmentation labels and radiomic features for all pre-operative multimodal magnetic resonance imaging (MRI) (n=243) of the multi-institutional glioma collections of The Cancer Genome Atlas (TCGA), publicly available in The Cancer Imaging Archive (TCIA). Pre-operative scans were identified in both glioblastoma (TCGA-GBM, n=135) and low-grade-glioma (TCGA-LGG, n=108) collections via radiological assessment. The glioma sub-region labels were produced by an automated state-of-the-art method and manually revised by an expert board-certified neuroradiologist. An extensive panel of radiomic features was extracted based on the manually-revised labels. This set of labels and features should enable i) direct utilization of the TCGA/TCIA glioma collections towards repeatable, reproducible and comparative quantitative studies leading to new predictive, prognostic, and diagnostic assessments, as well as ii) performance evaluation of computer-aided segmentation methods, and comparison to our state-of-the-art method. PMID:28872634

Zwitterionic polymer functionalization of polysulfone membrane with improved antifouling property and blood compatibility by combination of ATRP and click chemistry.

PubMed

Xiang, Tao; Lu, Ting; Xie, Yi; Zhao, Wei-Feng; Sun, Shu-Dong; Zhao, Chang-Sheng

2016-08-01

The chemical compositions are very important for designing blood-contacting membranes with good antifouling property and blood compatibility. In this study, we propose a method combining ATRP and click chemistry to introduce zwitterionic polymer of poly(sulfobetaine methacrylate) (PSBMA), negatively charged polymers of poly(sodium methacrylate) (PNaMAA) and/or poly(sodium p-styrene sulfonate) (PNaSS), to improve the antifouling property and blood compatibility of polysulfone (PSf) membranes. Attenuated total reflectance-Fourier transform infrared spectra, X-ray photoelectron spectroscopy and water contact angle results confirmed the successful grafting of the functional polymers. The antifouling property and blood compatibility of the modified membranes were systematically investigated. The zwitterionic polymer (PSBMA) grafted membranes showed good resistance to protein adsorption and bacterial adhesion; the negatively charged polymer (PNaSS or PNaMAA) grafted membranes showed improved blood compatibility, especially the anticoagulant property. Moreover, the PSBMA/PNaMAA modified membrane showed both antifouling property and anticoagulant property, and exhibited a synergistic effect in inhibiting blood coagulation. The functionalization of membrane surfaces by a combination of ATRP and click chemistry is demonstrated as an effective route to improve the antifouling property and blood compatibility of membranes in blood-contact. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Validity of the remote food photography method against doubly labeled water among minority preschoolers

USDA-ARS?s Scientific Manuscript database

The aim of this study was to determine the validity of energy intake (EI) estimations made using the remote food photography method (RFPM) compared to the doubly labeled water (DLW) method in minority preschool children in a free-living environment. Seven days of food intake and spot urine samples...

Fracture labelling of boar spermatozoa for the fucose-binding-protein (FBP).

PubMed

Friess, A E; Toepfer-Petersen, E; Schill, W B

1987-01-01

Labelling of fractured boar spermatozoa with the FUC-HRP gold method for a fucose-binding-protein (FBP) gave evidence the FBP is localized in the acrosomal matrix. All fracture faces through the acrosome from the rostral end towards the equatorial segment show similar labelling pattern. This labelling is completely blocked by preincubation of the fractured tissue with focoidan.

Combustion method for assay of biological materials labeled with carbon-14 or tritium, or double-labeled

NASA Technical Reports Server (NTRS)

Huebner, L. G.; Kisieleski, W. E.

1969-01-01

Dry catalytic combustion at high temperatures is used for assaying biological materials labeled carbon-14 and tritium, or double-labeled. A modified oxygen-flask technique is combined with standard vacuum-line techniques and includes convenience of direct in-vial collection of final combustion products, giving quantitative recovery of tritium and carbon-14.

Lyophilized Kit for the Preparation of the PET Perfusion Agent [(68)Ga]-MAA.

PubMed

Amor-Coarasa, Alejandro; Milera, Andrew; Carvajal, Denny; Gulec, Seza; McGoron, Anthony J

2014-01-01

Rapid developments in the field of medical imaging have opened new avenues for the use of positron emitting labeled microparticles. The radioisotope used in our research was (68)Ga, which is easy to obtain from a generator and has good nuclear properties for PET imaging. Methods. Commercially available macroaggregated albumin (MAA) microparticles were suspended in sterile saline, centrifuged to remove the free albumin and stannous chloride, relyophilized, and stored for later labeling with (68)Ga. Labeling was performed at different temperatures and times. (68)Ga purification settings were also tested and optimized. Labeling yield and purity of relyophilized MAA microparticles were compared with those that were not relyophilized. Results. MAA particles kept their original size distribution after relyophilization. Labeling yield was 98% at 75°C when a (68)Ga purification system was used, compared to 80% with unpurified (68)Ga. Radiochemical purity was over 97% up to 4 hours after the labeling. The relyophilized MAA and labeling method eliminate the need for centrifugation purification of the final product and simplify the labeling process. Animal experiments demonstrated the high in vivo stability of the obtained PET agent with more than 95% of the activity remaining in the lungs after 4 hours.

Performance evaluations of hybrid modulation with different optical labels over PDQ in high bit-rate OLS network systems.

PubMed

Xu, M; Li, Y; Kang, T Z; Zhang, T S; Ji, J H; Yang, S W

2016-11-14

Two orthogonal modulation optical label switching(OLS) schemes, which are based on payload of polarization multiplexing-differential quadrature phase shift keying(POLMUX-DQPSK or PDQ) modulated with identifications of duobinary (DB) label and pulse position modulation(PPM) label, are researched in high bit-rate OLS network. The BER performance of hybrid modulation with payload and label signals are discussed and evaluated in theory and simulation. The theoretical BER expressions of PDQ, PDQ-DB and PDQ-PPM are given with analysis method of hybrid modulation encoding in different the bit-rate ratios of payload and label. Theoretical derivation results are shown that the payload of hybrid modulation has a certain gain of receiver sensitivity than payload without label. The sizes of payload BER gain obtained from hybrid modulation are related to the different types of label. The simulation results are consistent with that of theoretical conclusions. The extinction ratio (ER) conflicting between hybrid encoding of intensity and phase types can be compromised and optimized in OLS system of hybrid modulation. The BER analysis method of hybrid modulation encoding in OLS system can be applied to other n-ary hybrid modulation or combination modulation systems.

Practical cell labeling with magnetite cationic liposomes for cell manipulation.

PubMed

Ito, Hiroshi; Nonogaki, Yurika; Kato, Ryuji; Honda, Hiroyuki

2010-07-01

Personalization of the cell culture process for cell therapy is an ideal strategy to obtain maximum treatment effects. In a previous report, we proposed a strategy using a magnetic manipulation device that combined a palm-top size device and a cell-labeling method using magnetite cationic liposomes (MCLs) to enable feasible personalized cell processing. In the present study, we focused on optimizing the MCL-labeling technique with respect to cell manipulation in small devices. From detailed analysis with different cell types, 4 pg/cell of MCL-label was found to be obtained immediately after mixing with MCLs, which was sufficient for magnetic cell manipulation. The amount of label increased within 24 h depending on cell type, although in all cases it decreased along with cell doubling, indicating that the labeling potential of MCLs was limited. The role of free MCLs not involved in labeling was also investigated; MCLs' role was found to be a supportive one that maximized the manipulation performance up to 100%. We also determined optimum conditions to manipulate adherent cells by MCL labeling using the MCL dispersed in trypsin solution. Considering labeling feasibility and practical performance with 10(3)-10(5) cells for personalized cell processing, we determined that 10 microg/ml of label without incubation time (0 h incubation) was the universal MCL-labeling condition. We propose the optimum specifications for a device to be combined with this method. 2010. Published by Elsevier B.V.

Method for locating metallic nitride inclusions in metallic alloy ingots

DOEpatents

White, Jack C.; Traut, Davis E.; Oden, Laurance L.; Schmitt, Roman A.

1992-01-01

A method of determining the location and history of metallic nitride and/or oxynitride inclusions in metallic melts. The method includes the steps of labeling metallic nitride and/or oxynitride inclusions by making a coreduced metallic-hafnium sponge from a mixture of hafnium chloride and the chloride of a metal, reducing the mixed chlorides with magnesium, nitriding the hafnium-labeled metallic-hafnium sponge, and seeding the sponge to be melted with hafnium-labeled nitride inclusions. The ingots are neutron activated and the hafnium is located by radiometric means. Hafnium possesses exactly the proper metallurgical and radiochemical properties for this use.

Factors associated with self-reported menu labeling use among US adults

PubMed Central

Lee-Kwan, Seung Hee; Pan, Liping; Maynard, Leah M.; McGuire, Lisa C.; Park, Sohyun

2016-01-01

Background Menu labeling may help people select foods and beverages with lower calories and is a potential population-based strategy to reduce obesity and diet-related chronic diseases in the United States. Objectives The aim of this cross-sectional study was to examine the prevalence of menu labeling use among adults and its association with sociodemographic, behavioral, and policy factors. Methods 2012 Behavioral Risk Factor Surveillance System data from 17 states that included 100,141 adults who noticed menu labeling at fast food/chain restaurants (“When calorie information is available in the restaurant, how often does this information help you decide what to order?”) were used. Menu labeling use was categorized: frequent (always/most of the time), moderate (half the time/sometimes), and never. Multinomial logistic regression was used to examine associations of sociodemographic, behavioral, and policy factors with menu labeling use. Results Overall, of adults who noticed menu labeling, 25.6% reported frequent use of menu labeling, 31.6% reported moderate use, and 42.7% reported that they never use menu labeling. Compared to never users, frequent users were significantly more likely to be younger, female, non-white, more educated, high-income, overweight or obese, physically active, former- or never-smokers, with no or lower (<1 time/day) sugar-sweetened beverage intake, and living in states where menu labeling legislation was enacted or proposed. Conclusions Menu labeling is one method that consumers can use to help reduce their calorie consumption from restaurants. These findings can be used to develop targeted interventions to increase menu labeling use among subpopulations with lower use. PMID:26875022

Assimilation efficiency for sediment-sorbed benzo(a)pyrene by Diporeia spp.

USGS Publications Warehouse

Lydy, M.J.; Landrum, P.F.

1993-01-01

Two methods are currently available for determining contaminant assimilation efficiencies (AE) from ingested material in benthic invertebrates. These methods were compared using the Great Lakes amphipod Diporeia spp. and [14C]benzo(a)pyrene (BaP) sorbed to Florissant sediment (< 63 µm). The first approach, the direct measurement method, uses total organic carbon as a tracer and yielded AE values ranging from 45.9~50.4%. The second approach, the dual-labeled method, uses 51Cr as a non-assimilated tracer and did not yield AE values for our data. The inability of the dual-labeled approach to estimate AEs was due, in part, to the selective feeding by Diporeia resulting in a failure of the non-assimilated tracer (51Cr) to track with the assimilated tracer ([14C]BaP). The failure of the dual-labeled approach was not a result of an uneven distribution of the labels among particle size classes, but more likely resulted from differential sorption of the two isotopically labeled materials to particles of differing composition. The [14C]BaP apparently sorbs to organic particles that are selectively ingested, while the 51Cr apparently sorbs to particles which are selectively excluded by Diporeia. The dual-labeled approach would be a viable and easier experimental approach for determining AE values if the characteristics that govern selective feeding can be determined.

Avoid violence, rioting, and outrage; approach celebration, delight, and strength: Using large text corpora to compute valence, arousal, and the basic emotions.

PubMed

Westbury, Chris; Keith, Jeff; Briesemeister, Benny B; Hofmann, Markus J; Jacobs, Arthur M

2015-01-01

Ever since Aristotle discussed the issue in Book II of his Rhetoric, humans have attempted to identify a set of "basic emotion labels". In this paper we propose an algorithmic method for evaluating sets of basic emotion labels that relies upon computed co-occurrence distances between words in a 12.7-billion-word corpus of unselected text from USENET discussion groups. Our method uses the relationship between human arousal and valence ratings collected for a large list of words, and the co-occurrence similarity between each word and emotion labels. We assess how well the words in each of 12 emotion label sets-proposed by various researchers over the past 118 years-predict the arousal and valence ratings on a test and validation dataset, each consisting of over 5970 items. We also assess how well these emotion labels predict lexical decision residuals (LDRTs), after co-varying out the effects attributable to basic lexical predictors. We then demonstrate a generalization of our method to determine the most predictive "basic" emotion labels from among all of the putative models of basic emotion that we considered. As well as contributing empirical data towards the development of a more rigorous definition of basic emotions, our method makes it possible to derive principled computational estimates of emotionality-specifically, of arousal and valence-for all words in the language.

Multi-location gram-positive and gram-negative bacterial protein subcellular localization using gene ontology and multi-label classifier ensemble.

PubMed

Wang, Xiao; Zhang, Jun; Li, Guo-Zheng

2015-01-01

It has become a very important and full of challenge task to predict bacterial protein subcellular locations using computational methods. Although there exist a lot of prediction methods for bacterial proteins, the majority of these methods can only deal with single-location proteins. But unfortunately many multi-location proteins are located in the bacterial cells. Moreover, multi-location proteins have special biological functions capable of helping the development of new drugs. So it is necessary to develop new computational methods for accurately predicting subcellular locations of multi-location bacterial proteins. In this article, two efficient multi-label predictors, Gpos-ECC-mPLoc and Gneg-ECC-mPLoc, are developed to predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. The two multi-label predictors construct the GO vectors by using the GO terms of homologous proteins of query proteins and then adopt a powerful multi-label ensemble classifier to make the final multi-label prediction. The two multi-label predictors have the following advantages: (1) they improve the prediction performance of multi-label proteins by taking the correlations among different labels into account; (2) they ensemble multiple CC classifiers and further generate better prediction results by ensemble learning; and (3) they construct the GO vectors by using the frequency of occurrences of GO terms in the typical homologous set instead of using 0/1 values. Experimental results show that Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently improve prediction accuracy of subcellular localization of multi-location gram-positive and gram-negative bacterial proteins respectively. The online web servers for Gpos-ECC-mPLoc and Gneg-ECC-mPLoc predictors are freely accessible at http://biomed.zzuli.edu.cn/bioinfo/gpos-ecc-mploc/ and http://biomed.zzuli.edu.cn/bioinfo/gneg-ecc-mploc/ respectively.

Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip.

PubMed

Wang, Chen; Ouyang, Jun; Ye, De-Kai; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

2012-08-07

Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.

OpenCL based machine learning labeling of biomedical datasets

NASA Astrophysics Data System (ADS)

Amoros, Oscar; Escalera, Sergio; Puig, Anna

2011-03-01

In this paper, we propose a two-stage labeling method of large biomedical datasets through a parallel approach in a single GPU. Diagnostic methods, structures volume measurements, and visualization systems are of major importance for surgery planning, intra-operative imaging and image-guided surgery. In all cases, to provide an automatic and interactive method to label or to tag different structures contained into input data becomes imperative. Several approaches to label or segment biomedical datasets has been proposed to discriminate different anatomical structures in an output tagged dataset. Among existing methods, supervised learning methods for segmentation have been devised to easily analyze biomedical datasets by a non-expert user. However, they still have some problems concerning practical application, such as slow learning and testing speeds. In addition, recent technological developments have led to widespread availability of multi-core CPUs and GPUs, as well as new software languages, such as NVIDIA's CUDA and OpenCL, allowing to apply parallel programming paradigms in conventional personal computers. Adaboost classifier is one of the most widely applied methods for labeling in the Machine Learning community. In a first stage, Adaboost trains a binary classifier from a set of pre-labeled samples described by a set of features. This binary classifier is defined as a weighted combination of weak classifiers. Each weak classifier is a simple decision function estimated on a single feature value. Then, at the testing stage, each weak classifier is independently applied on the features of a set of unlabeled samples. In this work, we propose an alternative representation of the Adaboost binary classifier. We use this proposed representation to define a new GPU-based parallelized Adaboost testing stage using OpenCL. We provide numerical experiments based on large available data sets and we compare our results to CPU-based strategies in terms of time and labeling speeds.

Improving condition severity classification with an efficient active learning based framework

PubMed Central

Nissim, Nir; Boland, Mary Regina; Tatonetti, Nicholas P.; Elovici, Yuval; Hripcsak, George; Shahar, Yuval; Moskovitch, Robert

2017-01-01

Classification of condition severity can be useful for discriminating among sets of conditions or phenotypes, for example when prioritizing patient care or for other healthcare purposes. Electronic Health Records (EHRs) represent a rich source of labeled information that can be harnessed for severity classification. The labeling of EHRs is expensive and in many cases requires employing professionals with high level of expertise. In this study, we demonstrate the use of Active Learning (AL) techniques to decrease expert labeling efforts. We employ three AL methods and demonstrate their ability to reduce labeling efforts while effectively discriminating condition severity. We incorporate three AL methods into a new framework based on the original CAESAR (Classification Approach for Extracting Severity Automatically from Electronic Health Records) framework to create the Active Learning Enhancement framework (CAESAR-ALE). We applied CAESAR-ALE to a dataset containing 516 conditions of varying severity levels that were manually labeled by seven experts. Our dataset, called the “CAESAR dataset,” was created from the medical records of 1.9 million patients treated at Columbia University Medical Center (CUMC). All three AL methods decreased labelers’ efforts compared to the learning methods applied by the original CAESER framework in which the classifier was trained on the entire set of conditions; depending on the AL strategy used in the current study, the reduction ranged from 48% to 64% that can result in significant savings, both in time and money. As for the PPV (precision) measure, CAESAR-ALE achieved more than 13% absolute improvement in the predictive capabilities of the framework when classifying conditions as severe. These results demonstrate the potential of AL methods to decrease the labeling efforts of medical experts, while increasing accuracy given the same (or even a smaller) number of acquired conditions. We also demonstrated that the methods included in the CAESAR-ALE framework (Exploitation and Combination_XA) are more robust to the use of human labelers with different levels of professional expertise. PMID:27016383

16 CFR 301.27 - Label and method of affixing.

Code of Federal Regulations, 2010 CFR

2010-01-01

... all times during the marketing of a fur product the required label shall have a minimum dimension of one and three-fourths (13/4) inches by two and three-fourths (23/4) inches (4.5 cm × 7 cm). Such label...

Simultaneous determination of ten Aconitum alkaloids in rat tissues by UHPLC-MS/MS and its application to a tissue distribution study on the compatibility of Heishunpian and Fritillariae thunbergii Bulbus.

PubMed

Yang, Bin; Xu, Yanyan; Wu, Yuanyuan; Wu, Huanyu; Wang, Yuan; Yuan, Lei; Xie, Jiabin; Li, Yubo; Zhang, Yanjun

2016-10-15

A rapid, sensitive and selective ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for simultaneous determination of ten Aconitum alkaloids in rat tissues. The tissue samples were prepared by a simple procedure protein precipitation with acetonitrile containing 0.1% acetic acid and separated on an Agilent XDB C18 column (4.6 mm×50mm, 1.8μm) using gradient elution with a mobile phase consisting of water and acetonitrile (both containing 0.1% formic acid) at a flow rate of 0.3mL/min. The quantitive determination was performed on an electrospray ionization (ESI) triple quadrupole tandem mass spectrometer using selective reaction monitoring (SRM) under positive ionization mode. The established method was fully validated according to the USA Food and Drug Administration (FDA) bioanalytical method validation guidance and the results demonstrated that the method was sensitive and selective with the lowest limits of quantification (LLOQ) at 0.025ng/mL in rat tissue homogenates. Meanwhile, the linearity, precision, accuracy, extraction recovery, matrix effect and stability were all within the required limits of biological sample analysis. After method validation, the validated method was successfully applied to the tissue distribution study on the compatibility of Heishunpian (HSP, the processed product of Aconitum carmichaelii Debx) and Fritillariae thunbergii Bulbus (Zhebeimu, ZBM). The results indicated that the distribution feature of monoester diterpenoid aconitines (MDAs), diester diterpenoid aconitines (DDAs) and non-ester alkaloids (NEAs) were inconsistency, and the compatibility of HSP and ZBM resulted in the distribution amount of DDAs increased in tissues. What's more, the results could provide the reliable basis for systematic research on the substance foundation of the compatibility of the herbal pair. Copyright © 2016 Elsevier B.V. All rights reserved.

Laminated and infused Parafilm® - paper for paper-based analytical devices.

PubMed

Kim, Yong Shin; Yang, Yuanyuan; Henry, Charles S

2018-02-01

Numerous fabrication methods have been reported for microfluidic paper-based analytical devices (μPADs) using barrier materials ranging from photoresist to wax. While these methods have been used with wide success, consistently producing small, high-resolution features using materials and methods that are compatible with solvents and surfactants remains a challenge. Two new methods are presented here for generating μPADs with well-defined, high-resolution structures compatible with solvents and surfactant-containing solutions by partially or fully fusing paper with Parafilm® followed by cutting with a CO 2 laser cutter. Partial fusion leads to laminated paper ( l -paper) while the complete fusion results in infused paper ( i -paper). Patterned structures in l -paper were fabricated by selective removal of the paper but not the underlying Parafilm® using a benchtop CO 2 laser. Under optimized conditions, a gap as small as 137 ± 22 μm could be generated. Using this approach, a miniaturized paper 384-zone plate, consisting of circular detection elements with a diameter of 1.86 mm, was fabricated in 64 × 43 mm 2 area. Furthermore, these ablation-patterned substrates were confirmed to be compatible with surfactant solutions and common organic solvents (methanol, acetonitrile and dimethylformamide), which has been achieved by very few μPAD patterning techniques. Patterns in i -paper were created by completely cutting out zones of the i -paper and then fixing pre-cut paper into these openings similar to the strategy of fitting a jigsaw piece into a puzzle. Upon heating, unmodified paper was readily sealed into these openings due to partial reflow of the paraffin into the paper. This unique and simple bonding method was illustrated by two types of 3D μPADs, a push-on valve and a time-gated flow distributor, without adding adhesive layers. The free-standing jigsaw-patterned sheets showed good structural stability and solution compatibility, which provided a facile alternative method for fabricating complicated μPADs.

Growth and Characterization of CuO Nanostructures on Si for the Fabrication of CuO/p-Si Schottky Diodes

PubMed Central

Çetinkaya, S.; Çetinkara, H. A.; Bayansal, F.; Kahraman, S.

2013-01-01

CuO interlayers in the CuO/p-Si Schottky diodes were fabricated by using CBD and sol-gel methods. Deposited CuO layers were characterized by SEM and XRD techniques. From the SEM images, it was seen that the film grown by CBD method is denser than the film grown by sol-gel method. This result is compatible with XRD results which show that the crystallization in CBD method is higher than it is in sol-gel method. For the electrical investigations, current-voltage characteristics of the diodes have been studied at room temperature. Conventional I-V and Norde's methods were used in order to determine the ideality factor, barrier height, and series resistance values. It was seen that the morphological and structural analysis are compatible with the results of electrical investigations. PMID:23766670

Stable Isotope Labeling Strategy for Curcumin Metabolite Study in Human Liver Microsomes by Liquid Chromatography-Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Gao, Dan; Chen, Xiaowu; Yang, Xiaomei; Wu, Qin; Jin, Feng; Wen, Hongliang; Jiang, Yuyang; Liu, Hongxia

2015-04-01

The identification of drug metabolites is very important in drug development. Nowadays, the most widely used methods are isotopes and mass spectrometry. However, the commercial isotopic labeled reagents are usually very expensive, and the rapid and convenient identification of metabolites is still difficult. In this paper, an 18O isotope labeling strategy was developed and the isotopes were used as a tool to identify drug metabolites using mass spectrometry. Curcumin was selected as a model drug to evaluate the established method, and the 18O labeled curcumin was successfully synthesized. The non-labeled and 18O labeled curcumin were simultaneously metabolized in human liver microsomes (HLMs) and analyzed by liquid chromatography/mass spectrometry (LC-MS). The two groups of chromatograms obtained from metabolic reaction mixture with and without cofactors were compared and analyzed using Metabolynx software (Waters Corp., Milford, MA, USA). The mass spectra of the newly appearing chromatographic peaks in the experimental sample were further analyzed to find the metabolite candidates. Their chemical structures were confirmed by tandem mass spectrometry. Three metabolites, including two reduction products and a glucuronide conjugate, were successfully detected under their specific HLMs metabolic conditions, which were in accordance with the literature reported results. The results demonstrated that the developed isotope labeling method, together with post-acquisition data processing using Metabolynx software, could be used for fast identification of new drug metabolites.

Engineering streptokinase for generation of active site-labeled plasminogen analogs*

PubMed Central

Laha, Malabika; Panizzi, Peter; Nahrendorf, Matthias; Bock, Paul E.

2011-01-01

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its non-proteolytic activation of the zymogen. The method is versatile and allows for stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH2-terminal His6-tags. The NH2-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile1 residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys414 residue and residues Arg253-Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm), and to disable Pg substrate binding to the SK·Pg*/Pm catalytic complexes, respectively. Near-elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His6 mutant increased the yield of labeled Pg 2.6-fold and reduced the time required >2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry. PMID:21570944

Evaluating the Impact of Menu Labeling on Food Choices and Intake

PubMed Central

Larsen, Peter D.; Agnew, Henry; Baik, Jenny; Brownell, Kelly D.

2010-01-01

Objectives. We assessed the impact of restaurant menu calorie labels on food choices and intake. Methods. Participants in a study dinner (n = 303) were randomly assigned to either (1) a menu without calorie labels (no calorie labels), (2) a menu with calorie labels (calorie labels), or (3) a menu with calorie labels and a label stating the recommended daily caloric intake for an average adult (calorie labels plus information). Food choices and intake during and after the study dinner were measured. Results. Participants in both calorie label conditions ordered fewer calories than those in the no calorie labels condition. When calorie label conditions were combined, that group consumed 14% fewer calories than the no calorie labels group. Individuals in the calorie labels condition consumed more calories after the study dinner than those in both other conditions. When calories consumed during and after the study dinner were combined, participants in the calorie labels plus information group consumed an average of 250 fewer calories than those in the other groups. Conclusions. Calorie labels on restaurant menus impacted food choices and intake; adding a recommended daily caloric requirement label increased this effect, suggesting menu label legislation should require such a label. Future research should evaluate menu labeling's impact on children's food choices and consumption. PMID:20019307

Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction

PubMed Central

Ban, Hitoshi; Nagano, Masanobu; Gavrilyuk, Julia; Hakamata, Wataru; Inokuma, Tsubasa; Barbas, Carlos F.

2013-01-01

The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective tri-functionalization of proteins is readily achieved. In particular cases, we noted PTAD decomposition resulted in formation of a putative isocyanate by-product that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene) glycol chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions. PMID:23534985

Understanding the compatibility of multiple uses on forest land: a survey of multiresource research with application to the Pacific Northwest.

Treesearch

James A. Stevens; Claire A. Montgomery

2002-01-01

In this report, multiresource research is described as it has coevolved with forest policy objectives—from managing for single or dominant uses, to managing for compatible multiple forest uses, to sustaining ecosystem health on the forest. The evolution of analytical methods for multiresource research is traced from impact analysis to multiresource modeling, and...

Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

PubMed Central

Soh, Nobuaki

2008-01-01

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology. PMID:27879749

Learning with imperfectly labeled patterns

NASA Technical Reports Server (NTRS)

Chittineni, C. B.

1979-01-01

The problem of learning in pattern recognition using imperfectly labeled patterns is considered. The performance of the Bayes and nearest neighbor classifiers with imperfect labels is discussed using a probabilistic model for the mislabeling of the training patterns. Schemes for training the classifier using both parametric and non parametric techniques are presented. Methods for the correction of imperfect labels were developed. To gain an understanding of the learning process, expressions are derived for success probability as a function of training time for a one dimensional increment error correction classifier with imperfect labels. Feature selection with imperfectly labeled patterns is described.

Single step signal group-imidazole labeling of organic phosphate groups under aqueous conditions

DOEpatents

Giese, R.W.; Wang, P.

1996-04-30

Compounds and methods for single step, covalent labeling of the phosphate group of an organic substance under aqueous conditions are described. The labeling compound includes any kind of detectable signal group covalently bound to an imidazole moiety, which can be imidazole or a substituted imidazole. A preferred labeling compound has the formula shown in the accompanying diagram. 4 figs.

[Application of DNA labeling technology in forensic botany].

PubMed

Znang, Xian; Li, Jing-Lin; Zhang, Xiang-Yu

2008-12-01

Forensic botany is a study of judicial plant evidence. Recently, researches on DNA labeling technology have been a mainstream of forensic botany. The article systematically reviews various types of DNA labeling techniques in forensic botany with enumerated practical cases, as well as the potential forensic application of each individual technique. The advantages of the DNA labeling technology over traditional morphological taxonomic methods are also summarized.

Multi-label classification of chronically ill patients with bag of words and supervised dimensionality reduction algorithms.

PubMed

Bromuri, Stefano; Zufferey, Damien; Hennebert, Jean; Schumacher, Michael

2014-10-01

This research is motivated by the issue of classifying illnesses of chronically ill patients for decision support in clinical settings. Our main objective is to propose multi-label classification of multivariate time series contained in medical records of chronically ill patients, by means of quantization methods, such as bag of words (BoW), and multi-label classification algorithms. Our second objective is to compare supervised dimensionality reduction techniques to state-of-the-art multi-label classification algorithms. The hypothesis is that kernel methods and locality preserving projections make such algorithms good candidates to study multi-label medical time series. We combine BoW and supervised dimensionality reduction algorithms to perform multi-label classification on health records of chronically ill patients. The considered algorithms are compared with state-of-the-art multi-label classifiers in two real world datasets. Portavita dataset contains 525 diabetes type 2 (DT2) patients, with co-morbidities of DT2 such as hypertension, dyslipidemia, and microvascular or macrovascular issues. MIMIC II dataset contains 2635 patients affected by thyroid disease, diabetes mellitus, lipoid metabolism disease, fluid electrolyte disease, hypertensive disease, thrombosis, hypotension, chronic obstructive pulmonary disease (COPD), liver disease and kidney disease. The algorithms are evaluated using multi-label evaluation metrics such as hamming loss, one error, coverage, ranking loss, and average precision. Non-linear dimensionality reduction approaches behave well on medical time series quantized using the BoW algorithm, with results comparable to state-of-the-art multi-label classification algorithms. Chaining the projected features has a positive impact on the performance of the algorithm with respect to pure binary relevance approaches. The evaluation highlights the feasibility of representing medical health records using the BoW for multi-label classification tasks. The study also highlights that dimensionality reduction algorithms based on kernel methods, locality preserving projections or both are good candidates to deal with multi-label classification tasks in medical time series with many missing values and high label density. Copyright © 2014 Elsevier Inc. All rights reserved.

Proteomic detection of oxidized and reduced thiol proteins in cultured cells.

PubMed

Cuddihy, Sarah L; Baty, James W; Brown, Kristin K; Winterbourn, Christine C; Hampton, Mark B

2009-01-01

The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

7 CFR 60.300 - Labeling.

Code of Federal Regulations, 2012 CFR

2012-01-01

... AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) COUNTRY OF ORIGIN LABELING FOR FISH AND SHELLFISH General Provisions Country of Origin Notification § 60.300 Labeling. (a) Country of origin declarations and method of production (wild and/or farm-raised) designations can either...

7 CFR 60.300 - Labeling.

Code of Federal Regulations, 2014 CFR

2014-01-01

... AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) COUNTRY OF ORIGIN LABELING FOR FISH AND SHELLFISH General Provisions Country of Origin Notification § 60.300 Labeling. (a) Country of origin declarations and method of production (wild and/or farm-raised) designations can either...

7 CFR 60.300 - Labeling.

Code of Federal Regulations, 2013 CFR

2013-01-01

... AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) COUNTRY OF ORIGIN LABELING FOR FISH AND SHELLFISH General Provisions Country of Origin Notification § 60.300 Labeling. (a) Country of origin declarations and method of production (wild and/or farm-raised) designations can either...

7 CFR 60.300 - Labeling.

Code of Federal Regulations, 2011 CFR

2011-01-01

... AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) COUNTRY OF ORIGIN LABELING FOR FISH AND SHELLFISH General Provisions Country of Origin Notification § 60.300 Labeling. (a) Country of origin declarations and method of production (wild and/or farm-raised) designations can either...

Detection of viral genomes in the liver by in situ hybridization using 35S-, bromodeoxyuridine-, and biotin-labeled probes

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

Niedobitek, G.; Finn, T.; Herbst, H.

1989-03-01

Methods employing /sup 35/S-, biotin-, and bromodeoxyuridine (BrdUrd)-labeled DNA probes were compared for the detection of hepatitis B virus (HBV) and cytomegalovirus (CMV) in the liver. The results demonstrate that: 1) HBV can be detected reliably only by the use of radiolabeled probes, whereas methods employing nonradioactive probes obviously are not sensitive enough for this virus. The use of /sup 35/S-labeled probes shortens the exposure times considerably in comparison to tritiated probes. 2) Biotin-labeled probes are of limited value for in situ hybridization on liver tissues because the presence of endogenous avidin-binding activity often leads to false positive results. 3)more » Brd-Urd-labeled probes are a useful alternative to biotinylated probes for the detection of CMV. In comparison with biotinylated probes, BrdUrd-labeled probes produce a specific signal of similar staining intensity in the absence of background staining in the liver.« less