Characterization of a novel particle into liquid sampler for analysis of single fluorescent aerosol particles through capillary electrophoresis.

PubMed

Tang, Hao; Hiemstra, Scott; Thompson, Jonathan E

2011-09-19

An approach to sample and analyze single aerosolized droplets (<10 nL) of solutions containing fluorescein isothiocyanate (FITC) labeled glycine (GLY) and glutamic acid (GLU) is demonstrated. The sampling approach is based on inertial impaction in which the sample particle is accelerated through a nozzle and directly into a small drop of buffered solution (20 mM borate, pH=10) suspended at the end of a coaxial tube of stainless steel and a fused silica capillary. A spherical light scattering cell and laser (λ=532 nm) is used to detect the arrival of particles at the buffered droplet. Upon dissolution and/or mixing, a portion of the sample is injected onto the fused silica capillary for subsequent chemical analysis by capillary electrophoresis (CE) and detection by laser-induced fluorescence (LIF). It was found that the inertial impaction approach sampled particles >1 μm diameter with an efficiency of 80% or greater. At 15 kV applied potential, the FITC conjugates of GLY and GLU could be resolved in less than 120 s allowing qualitative analysis of the contents of single dispersed particles. However, the extent to which the sample is diluted into the buffer droplet varied significantly on a per-particle basis that caused >80% R.S.D. in fluorescence peak heights. This aspect of the method would necessitate the use of internal standards for quantitative analysis of materials present within the particles. It is envisaged that further improvements to the device described may ultimately lead to analysis of the contents of single particles dispersed in earth's atmosphere. Copyright © 2011 Elsevier B.V. All rights reserved.

Stable isotope dilution analysis of hydrologic samples by inductively coupled plasma mass spectrometry

USGS Publications Warehouse

Garbarino, John R.; Taylor, Howard E.

1987-01-01

Inductively coupled plasma mass spectrometry is employed in the determination of Ni, Cu, Sr, Cd, Ba, Ti, and Pb in nonsaline, natural water samples by stable isotope dilution analysis. Hydrologic samples were directly analyzed without any unusual pretreatment. Interference effects related to overlapping isobars, formation of metal oxide and multiply charged ions, and matrix composition were identified and suitable methods of correction evaluated. A comparability study snowed that single-element isotope dilution analysis was only marginally better than sequential multielement isotope dilution analysis. Accuracy and precision of the single-element method were determined on the basis of results obtained for standard reference materials. The instrumental technique was shown to be ideally suited for programs associated with certification of standard reference materials.

Laser dissection sampling modes for direct mass spectral analysis [using a hybrid optical microscopy/laser ablation liquid vortex capture/electrospray ionization system

DOE PAGES

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

2016-02-01

Here, laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis ofmore » single cells and tissue. Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (4-15 m) even when agglomerated together. Turbid Allium Cepa cells (150 m) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. In conclusion, laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.« less

Laser dissection sampling modes for direct mass spectral analysis [using a hybrid optical microscopy/laser ablation liquid vortex capture/electrospray ionization system

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

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

Here, laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis ofmore » single cells and tissue. Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (4-15 m) even when agglomerated together. Turbid Allium Cepa cells (150 m) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. In conclusion, laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.« less

ESTIMATING SYSTEMIC EXPOSURE TO ETHINYL ESTRADIOL FROM AN ORAL CONTRACEPTIVE

PubMed Central

WESTHOFF, Carolyn L.; PIKE, Malcolm C.; TANG, Rosalind; DINAPOLI, Marianne N.; SULL, Monica; CREMERS, Serge

2015-01-01

Objectives This study was conducted to compare single-dose pharmacokinetics of ethinyl estradiol in an oral contraceptive to steady-state values, and to assess whether any simpler measures could provide an adequate proxy of the ‘gold standard’ 24-hour steady-state area-under-the-curve. Identifying a simple, less expensive, measure of systemic ethinyl estradiol exposure would be useful for larger studies designed to assess the relationship between an individual’s ethinyl estradiol exposure and her side effects. Study Design We conducted a 13 samples over 24 hours pharmacokinetic analysis on day 1 and day 21 of the first cycle of a monophasic oral contraceptive containing 30 mcg ethinyl estradiol and 150 mcg levonorgestrel in 17 non-obese healthy white women. We also conducted an abbreviated single dose 9-sample pharmacokinetic analysis after a month washout. Ethinyl estradiol was measured by liquid chromatography-tandem mass spectrometry. We compared results of full 13-sample steady-state pharmacokinetic analysis with results calculated using fewer samples (9 or 5) and following the single doses. We calculated Pearson correlation coefficients to evaluate the relationships between these estimates of systemic ethinyl estradiol exposure. Results The area-under-the-curve, maximum (Cmax), and 24-hour (C24) values were similar following the two single oral contraceptive doses (area-under-the-curve, r = 0.92). The steady-state 13-sample 24-hour area-under-the-curve was highly correlated with the average 9-sample area-under-the-curve after the two single doses (r = 0.81, p = 0.0002). This correlation remained the same if the number of samples was reduced to 4, taken at time 1, 2.5, 4 and 24 hours. The C24 at steady-state was highly correlated with the 24-hour steady-state area-under-the-curve (r = 0.92, p < 0.0001). The average of the C24 values following the two single doses was also quite highly correlated with the steady-state area-under-the-curve (r = 0.72, p = 0.0026). Conclusions Limited blood sampling, including results from two single doses, gave highly correlated estimates of an oral contraceptive user’s steady-state ethinyl estradiol exposure. PMID:25511238

Thermogravimetric Analysis of Single-Wall Carbon Nanotubes

NASA Technical Reports Server (NTRS)

Arepalli, Sivram; Nikolaev, Pavel; Gorelik, Olga

2010-01-01

An improved protocol for thermogravimetric analysis (TGA) of samples of single-wall carbon nanotube (SWCNT) material has been developed to increase the degree of consistency among results so that meaningful comparisons can be made among different samples. This improved TGA protocol is suitable for incorporation into the protocol for characterization of carbon nanotube material. In most cases, TGA of carbon nanotube materials is performed in gas mixtures that contain oxygen at various concentrations. The improved protocol is summarized.

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

NASA Astrophysics Data System (ADS)

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Qiang, Yu; Liu, Ke; Cui, Chengjun; Chen, Jianhua; Huang, Qing; Hu, Jun; Wang, Lianhui; Huang, Wei; Shi, Yongyong; Fan, Chunhai

2017-04-01

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ~10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level.

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

PubMed Central

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Qiang, Yu; Liu, Ke; Cui, Chengjun; Chen, Jianhua; Huang, Qing; Hu, Jun; Wang, Lianhui; Huang, Wei; Shi, Yongyong; Fan, Chunhai

2017-01-01

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ∼10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level. PMID:28382928

Segmental hair analysis for differentiation of tilidine intake from external contamination using LC-ESI-MS/MS and MALDI-MS/MS imaging.

PubMed

Poetzsch, Michael; Baumgartner, Markus R; Steuer, Andrea E; Kraemer, Thomas

2015-02-01

Segmental hair analysis has been used for monitoring changes of consumption habit of drugs. Contamination from the environment or sweat might cause interpretative problems. For this reason, hair analysis results were compared in hair samples taken 24 h and 30 days after a single tilidine dose. The 24-h hair samples already showed high concentrations of tilidine and nortilidine. Analysis of wash water from sample preparation confirmed external contamination by sweat as reason. The 30-day hair samples were still positive for tilidine in all segments. Negative wash-water analysis proved incorporation from sweat into the hair matrix. Interpretation of a forensic case was requested where two children had been administered tilidine by their nanny and tilidine/nortilidine had been detected in all hair segments, possibly indicating multiple applications. Taking into consideration the results of the present study and of MALDI-MS imaging, a single application as cause for analytical results could no longer be excluded. Interpretation of consumption behaviour of tilidine based on segmental hair analysis has to be done with caution, even after typical wash procedures during sample preparation. External sweat contamination followed by incorporation into the hair matrix can mimic chronic intake. For assessment of external contamination, hair samples should not only be collected several weeks but also one to a few days after intake. MALDI-MS imaging of single hair can be a complementary tool for interpretation. Limitations for interpretation of segmental hair analysis shown here might also be applicable to drugs with comparable physicochemical and pharmacokinetic properties. Copyright © 2014 John Wiley & Sons, Ltd.

Heterogeneity of Metazoan Cells and Beyond: To Integrative Analysis of Cellular Populations at Single-Cell Level.

PubMed

Barteneva, Natasha S; Vorobjev, Ivan A

2018-01-01

In this paper, we review some of the recent advances in cellular heterogeneity and single-cell analysis methods. In modern research of cellular heterogeneity, there are four major approaches: analysis of pooled samples, single-cell analysis, high-throughput single-cell analysis, and lately integrated analysis of cellular population at a single-cell level. Recently developed high-throughput single-cell genetic analysis methods such as RNA-Seq require purification step and destruction of an analyzed cell often are providing a snapshot of the investigated cell without spatiotemporal context. Correlative analysis of multiparameter morphological, functional, and molecular information is important for differentiation of more uniform groups in the spectrum of different cell types. Simplified distributions (histograms and 2D plots) can underrepresent biologically significant subpopulations. Future directions may include the development of nondestructive methods for dissecting molecular events in intact cells, simultaneous correlative cellular analysis of phenotypic and molecular features by hybrid technologies such as imaging flow cytometry, and further progress in supervised and non-supervised statistical analysis algorithms.

Sample flow switching techniques on microfluidic chips.

PubMed

Pan, Yu-Jen; Lin, Jin-Jie; Luo, Win-Jet; Yang, Ruey-Jen

2006-02-15

This paper presents an experimental investigation into electrokinetically focused flow injection for bio-analytical applications. A novel microfluidic device for microfluidic sample handling is presented. The microfluidic chip is fabricated on glass substrates using conventional photolithographic and chemical etching processes and is bonded using a high-temperature fusion method. The proposed valve-less device is capable not only of directing a single sample flow to a specified output port, but also of driving multiple samples to separate outlet channels or even to a single outlet to facilitate sample mixing. The experimental results confirm that the sample flow can be electrokinetically pre-focused into a narrow stream and guided to the desired outlet port by means of a simple control voltage model. The microchip presented within this paper has considerable potential for use in a variety of applications, including high-throughput chemical analysis, cell fusion, fraction collection, sample mixing, and many other applications within the micro-total-analysis systems field.

Nanohole optical tweezers in heterogeneous mixture analysis

NASA Astrophysics Data System (ADS)

Hacohen, Noa; Ip, Candice J. X.; Laxminarayana, Gurunatha K.; DeWolf, Timothy S.; Gordon, Reuven

2017-08-01

Nanohole optical trapping is a tool that has been shown to analyze proteins at the single molecule level using pure samples. The next step is to detect and study single molecules with dirty samples. We demonstrate that using our double nanohole optical tweezing configuration, single particles in an egg white solution can be classified when trapped. Different sized molecules provide different signal variations in their trapped state, allowing the proteins to be statistically characterized. Root mean squared variation and trap stiffness are methods used on trapped signals to distinguish between the different proteins. This method to isolate and determine single molecules in heterogeneous samples provides huge potential to become a reliable tool for use within biomedical and scientific communities.

Development of a Multiplex Single Base Extension Assay for Mitochondrial DNA Haplogroup Typing

PubMed Central

Nelson, Tahnee M.; Just, Rebecca S.; Loreille, Odile; Schanfield, Moses S.; Podini, Daniele

2007-01-01

Aim To provide a screening tool to reduce time and sample consumption when attempting mtDNA haplogroup typing. Methods A single base primer extension assay was developed to enable typing, in a single reaction, of twelve mtDNA haplogroup specific polymorphisms. For validation purposes a total of 147 samples were tested including 73 samples successfully haplogroup typed using mtDNA control region (CR) sequence data, 21 samples inconclusively haplogroup typed by CR data, 20 samples previously haplogroup typed using restriction fragment length polymorphism (RFLP) analysis, and 31 samples of known ancestral origin without previous haplogroup typing. Additionally, two highly degraded human bones embalmed and buried in the early 1950s were analyzed using the single nucleotide polymorphisms (SNP) multiplex. Results When the SNP multiplex was used to type the 96 previously CR sequenced specimens, an increase in haplogroup or macrohaplogroup assignment relative to conventional CR sequence analysis was observed. The single base extension assay was also successfully used to assign a haplogroup to decades-old, embalmed skeletal remains dating to World War II. Conclusion The SNP multiplex was successfully used to obtain haplogroup status of highly degraded human bones, and demonstrated the ability to eliminate possible contributors. The SNP multiplex provides a low-cost, high throughput method for typing of mtDNA haplogroups A, B, C, D, E, F, G, H, L1/L2, L3, M, and N that could be useful for screening purposes for human identification efforts and anthropological studies. PMID:17696300

Selective counting and sizing of single virus particles using fluorescent aptamer-based nanoparticle tracking analysis.

PubMed

Szakács, Zoltán; Mészáros, Tamás; de Jonge, Marien I; Gyurcsányi, Róbert E

2018-05-30

Detection and counting of single virus particles in liquid samples are largely limited to narrow size distribution of viruses and purified formulations. To address these limitations, here we propose a calibration-free method that enables concurrently the selective recognition, counting and sizing of virus particles as demonstrated through the detection of human respiratory syncytial virus (RSV), an enveloped virus with a broad size distribution, in throat swab samples. RSV viruses were selectively labeled through their attachment glycoproteins (G) with fluorescent aptamers, which further enabled their identification, sizing and counting at the single particle level by fluorescent nanoparticle tracking analysis. The proposed approach seems to be generally applicable to virus detection and quantification. Moreover, it could be successfully applied to detect single RSV particles in swab samples of diagnostic relevance. Since the selective recognition is associated with the sizing of each detected particle, this method enables to discriminate viral elements linked to the virus as well as various virus forms and associations.

Genetic Association of MPPED2 and ACTN2 with Dental Caries

PubMed Central

Stanley, B.O.C.; Feingold, E.; Cooper, M.; Vanyukov, M.M.; Maher, B.S.; Slayton, R.L.; Willing, M.C.; Reis, S.E.; McNeil, D.W.; Crout, R.J.; Weyant, R.J.; Levy, S.M.; Vieira, A.R.; Marazita, M.L.; Shaffer, J.R.

2014-01-01

The first genome-wide association study of dental caries focused on primary teeth in children aged 3 to 12 yr and nominated several novel genes: ACTN2, EDARADD, EPHA7, LPO, MPPED2, MTR, and ZMPSTE24. Here we interrogated 156 single-nucleotide polymorphisms (SNPs) within these candidate genes for evidence of association with dental caries experience in 13 race- and age-stratified samples from 6 independent studies (n = 3600). Analysis was performed separately for each sample, and results were combined across samples via meta-analysis. MPPED2 was significantly associated with caries via meta-analysis across the 5 childhood samples, with 4 SNPs showing significant associations after gene-wise adjustment for multiple comparisons (p < .0026). These results corroborate the previous genome-wide association study, although the functional role of MPPED2 in caries etiology remains unknown. ACTN2 also showed significant association via meta-analysis across childhood samples (p = .0014). Moreover, in adults, genetic association was observed for ACTN2 SNPs in individual samples (p < .0025), but no single SNP was significant via meta-analysis across all 8 adult samples. Given its compelling biological role in organizing ameloblasts during amelogenesis, this study strengthens the hypothesis that ACTN2 influences caries risk. Results for the other candidate genes neither proved nor precluded their associations with dental caries. PMID:24810274

Using dried blood spot sampling to improve data quality and reduce animal use in mouse pharmacokinetic studies.

PubMed

Wickremsinhe, Enaksha R; Perkins, Everett J

2015-03-01

Traditional pharmacokinetic analysis in nonclinical studies is based on the concentration of a test compound in plasma and requires approximately 100 to 200 μL blood collected per time point. However, the total blood volume of mice limits the number of samples that can be collected from an individual animal-often to a single collection per mouse-thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a sparse-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to 20 μL), thus supporting serial blood sampling and the generation of a complete pharmacokinetic profile from a single mouse. Here we compare plasma-derived data with DBS-derived data, explain how to adopt DBS sampling to support discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that use DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a single study.

Using Dried Blood Spot Sampling to Improve Data Quality and Reduce Animal Use in Mouse Pharmacokinetic Studies

PubMed Central

Wickremsinhe, Enaksha R; Perkins, Everett J

2015-01-01

Traditional pharmacokinetic analysis in nonclinical studies is based on the concentration of a test compound in plasma and requires approximately 100 to 200 µL blood collected per time point. However, the total blood volume of mice limits the number of samples that can be collected from an individual animal—often to a single collection per mouse—thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a sparse-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to 20 µL), thus supporting serial blood sampling and the generation of a complete pharmacokinetic profile from a single mouse. Here we compare plasma-derived data with DBS-derived data, explain how to adopt DBS sampling to support discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that use DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a single study. PMID:25836959

Computerized EEG analysis for studying the effect of drugs on the central nervous system.

PubMed

Rosadini, G; Cavazza, B; Rodriguez, G; Sannita, W G; Siccardi, A

1977-11-01

Samples of our experience in quantitative pharmaco-EEG are reviewed to discuss and define its applicability and limits. Simple processing systems, such as the computation of Hjorth's descriptors, are useful for on-line monitoring of drug-induced EEG modifications which are evident also at the visual visual analysis. Power spectral analysis is suitable to identify and quantify EEG effects not evident at the visual inspection. It demonstrated how the EEG effects of compounds in a long-acting formulation vary according to the sampling time and the explored cerebral area. EEG modifications not detected by power spectral analysis can be defined by comparing statistically (F test) the spectral values of the EEG from a single lead at the different samples (longitudinal comparison), or the spectral values from different leads at any sample (intrahemispheric comparison). The presently available procedures of quantitative pharmaco-EEG are effective when applied to the study of mutltilead EEG recordings in a statistically significant sample of population. They do not seem reliable in the monitoring of directing of neuropyschiatric therapies in single patients, due to individual variability of drug effects.

Proteomic analysis of formalin-fixed paraffin embedded tissue by MALDI imaging mass spectrometry

PubMed Central

Casadonte, Rita; Caprioli, Richard M

2012-01-01

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE -TMAs using matrix-assisted laser desorption/ionization (MAL DI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE -TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis. PMID:22011652

Using Single Drop Microextraction for Headspace Analysis with Gas Chromatography

NASA Astrophysics Data System (ADS)

Riccio, Daniel; Wood, Derrick C.; Miller, James M.

2008-07-01

Headspace (HS) gas chromatography (GC) is commonly used to analyze samples that contain non-volatiles. In 1996, a new sampling technique called single drop microextraction, SDME, was introduced, and in 2001 it was applied to HS analysis. It is a simple technique that uses equipment normally found in the undergraduate laboratory, making it ideal for instructional use, especially to illustrate HS analysis or as an alternative to solid-phase microextraction (SPME) to which it is very similar. The basic principles and practice of HS-GC using SDME are described, including a complete review of the literature. Some possible experiments are suggested using water and N -methylpyrrolidone (NMP) as solvents.

Combined target factor analysis and Bayesian soft-classification of interference-contaminated samples: forensic fire debris analysis.

PubMed

Williams, Mary R; Sigman, Michael E; Lewis, Jennifer; Pitan, Kelly McHugh

2012-10-10

A bayesian soft classification method combined with target factor analysis (TFA) is described and tested for the analysis of fire debris data. The method relies on analysis of the average mass spectrum across the chromatographic profile (i.e., the total ion spectrum, TIS) from multiple samples taken from a single fire scene. A library of TIS from reference ignitable liquids with assigned ASTM classification is used as the target factors in TFA. The class-conditional distributions of correlations between the target and predicted factors for each ASTM class are represented by kernel functions and analyzed by bayesian decision theory. The soft classification approach assists in assessing the probability that ignitable liquid residue from a specific ASTM E1618 class, is present in a set of samples from a single fire scene, even in the presence of unspecified background contributions from pyrolysis products. The method is demonstrated with sample data sets and then tested on laboratory-scale burn data and large-scale field test burns. The overall performance achieved in laboratory and field test of the method is approximately 80% correct classification of fire debris samples. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Headspace Single-Drop Microextraction Gas Chromatography Mass Spectrometry for the Analysis of Volatile Compounds from Herba Asari

PubMed Central

Wang, Guan-Jie; Tian, Li; Fan, Yu-Ming; Qi, Mei-Ling

2013-01-01

A rapid headspace single-drop microextraction gas chromatography mass spectrometry (SDME-GC-MS) for the analysis of the volatile compounds in Herba Asari was developed in this study. The extraction solvent, extraction temperature and time, sample amount, and particle size were optimized. A mixed solvent of n-tridecane and butyl acetate (1 : 1) was finally used for the extraction with sample amount of 0.750 g and 100-mesh particle size at 70°C for 15 min. Under the determined conditions, the pound samples of Herba Asari were directly applied for the analysis. The result showed that SDME-GC–MS method was a simple, effective, and inexpensive way to measure the volatile compounds in Herba Asari and could be used for the analysis of volatile compounds in Chinese medicine. PMID:23607049

NHEXAS PHASE I MARYLAND STUDY--STANDARD OPERATING PROCEDURE FOR COLLECTION, STORAGE, AND SHIPMENT OF BLOOD SAMPLES FOR METAL, PESTICIDE, PAH, VOC, AND LIPID ANALYSIS (F11)

EPA Science Inventory

The purpose of this SOP is to describe collection, storage, and shipment requirements of blood samples for metal, pesticide, polynuclear aromatic hydrocarbons (PAHs), volatile organic compound (VOC), and lipid analysis. Seven samples were taken from a single puncture: two 3-mL t...

Clonal architecture of secondary acute myeloid leukemia defined by single-cell sequencing.

PubMed

Hughes, Andrew E O; Magrini, Vincent; Demeter, Ryan; Miller, Christopher A; Fulton, Robert; Fulton, Lucinda L; Eades, William C; Elliott, Kevin; Heath, Sharon; Westervelt, Peter; Ding, Li; Conrad, Donald F; White, Brian S; Shao, Jin; Link, Daniel C; DiPersio, John F; Mardis, Elaine R; Wilson, Richard K; Ley, Timothy J; Walter, Matthew J; Graubert, Timothy A

2014-07-01

Next-generation sequencing has been used to infer the clonality of heterogeneous tumor samples. These analyses yield specific predictions-the population frequency of individual clones, their genetic composition, and their evolutionary relationships-which we set out to test by sequencing individual cells from three subjects diagnosed with secondary acute myeloid leukemia, each of whom had been previously characterized by whole genome sequencing of unfractionated tumor samples. Single-cell mutation profiling strongly supported the clonal architecture implied by the analysis of bulk material. In addition, it resolved the clonal assignment of single nucleotide variants that had been initially ambiguous and identified areas of previously unappreciated complexity. Accordingly, we find that many of the key assumptions underlying the analysis of tumor clonality by deep sequencing of unfractionated material are valid. Furthermore, we illustrate a single-cell sequencing strategy for interrogating the clonal relationships among known variants that is cost-effective, scalable, and adaptable to the analysis of both hematopoietic and solid tumors, or any heterogeneous population of cells.

Dielectrophoretic Capture and Genetic Analysis of Single Neuroblastoma Tumor Cells

PubMed Central

Carpenter, Erica L.; Rader, JulieAnn; Ruden, Jacob; Rappaport, Eric F.; Hunter, Kristen N.; Hallberg, Paul L.; Krytska, Kate; O’Dwyer, Peter J.; Mosse, Yael P.

2014-01-01

Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients. PMID:25133137

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

Not Available

This volume contains the interim change notice for sample preparation methods. Covered are: acid digestion for metals analysis, fusion of Hanford tank waste solids, water leach of sludges/soils/other solids, extraction procedure toxicity (simulate leach in landfill), sample preparation for gamma spectroscopy, acid digestion for radiochemical analysis, leach preparation of solids for free cyanide analysis, aqueous leach of solids for anion analysis, microwave digestion of glasses and slurries for ICP/MS, toxicity characteristic leaching extraction for inorganics, leach/dissolution of activated metal for radiochemical analysis, extraction of single-shell tank (SST) samples for semi-VOC analysis, preparation and cleanup of hydrocarbon- containing samples for VOCmore » and semi-VOC analysis, receiving of waste tank samples in onsite transfer cask, receipt and inspection of SST samples, receipt and extrusion of core samples at 325A shielded facility, cleaning and shipping of waste tank samplers, homogenization of solutions/slurries/sludges, and test sample preparation for bioassay quality control program.« less

Analysis of SPAR 8 single-axis levitation experiment

NASA Technical Reports Server (NTRS)

Rush, J. E.; Schafer, C. F.; Holland, R. L.

1981-01-01

The melting and resolidification of SPAR 8 payload melting and resolidification of a glass specimen from the in a containerless condition and the retrieval and examination of the specimen from the. The absence of container contact was assured by use of a single-axis acoustic levitation system. However, the sample contacted a wire cage after being held without container contact by the acoustic field for only approximately 87 seconds. At this time, the sample was still molten and, therefore, flowed aroung the wire and continued to adhere to it. An analysis of why the sample did not remain levitated free of container contact is presented. The experiment is described, and experimental observations are discussed and analyzed.

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence

PubMed Central

Farash, Katherine; Hanson, Erin K.; Ballantyne, Jack

2015-01-01

DNA profiles can be obtained from ‘touch DNA’ evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a ‘blind-swabbing’ approach will co-sample cellular material from the different individuals, even if the individuals’ cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim’s DNA may be found in significant excess thus masking any potential perpetrator’s DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, ‘smart analysis’ method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., “clumps”) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material. PMID:25867046

Design and Analysis of Single-Cell Sequencing Experiments.

PubMed

Grün, Dominic; van Oudenaarden, Alexander

2015-11-05

Recent advances in single-cell sequencing hold great potential for exploring biological systems with unprecedented resolution. Sequencing the genome of individual cells can reveal somatic mutations and allows the investigation of clonal dynamics. Single-cell transcriptome sequencing can elucidate the cell type composition of a sample. However, single-cell sequencing comes with major technical challenges and yields complex data output. In this Primer, we provide an overview of available methods and discuss experimental design and single-cell data analysis. We hope that these guidelines will enable a growing number of researchers to leverage the power of single-cell sequencing. Copyright © 2015 Elsevier Inc. All rights reserved.

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

EPA Science Inventory

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

Numerical study of ultra-low field nuclear magnetic resonance relaxometry utilizing a single axis magnetometer for signal detection.

PubMed

Vogel, Michael W; Vegh, Viktor; Reutens, David C

2013-05-01

This paper investigates optimal placement of a localized single-axis magnetometer for ultralow field (ULF) relaxometry in view of various sample shapes and sizes. The authors used finite element method for the numerical analysis to determine the sample magnetic field environment and evaluate the optimal location of the single-axis magnetometer. Given the different samples, the authors analysed the magnetic field distribution around the sample and determined the optimal orientation and possible positions of the sensor to maximize signal strength, that is, the power of the free induction decay. The authors demonstrate that a glass vial with flat bottom and 10 ml volume is the best structure to achieve the highest signal out of samples studied. This paper demonstrates the importance of taking into account the combined effects of sensor configuration and sample parameters for signal generation prior to designing and constructing ULF systems with a single-axis magnetometer. Through numerical simulations the authors were able to optimize structural parameters, such as sample shape and size, sensor orientation and location, to maximize the measured signal in ultralow field relaxometry.

Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis.

PubMed

Jakupciak, John P; Wells, Jeffrey M; Karalus, Richard J; Pawlowski, David R; Lin, Jeffrey S; Feldman, Andrew B

2013-01-01

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.

Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis

PubMed Central

Jakupciak, John P.; Wells, Jeffrey M.; Karalus, Richard J.; Pawlowski, David R.; Lin, Jeffrey S.; Feldman, Andrew B.

2013-01-01

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations. PMID:24455204

Noninvasive metabolic profiling using microfluidics for analysis of single preimplantation embryos.

PubMed

Urbanski, John Paul; Johnson, Mark T; Craig, David D; Potter, David L; Gardner, David K; Thorsen, Todd

2008-09-01

Noninvasive analysis of metabolism at the single cell level will have many applications in evaluating cellular physiology. One clinically relevant application would be to determine the metabolic activities of embryos produced through assisted reproduction. There is increasing evidence that embryos with greater developmental capacity have distinct metabolic profiles. One of the standard techniques for evaluating embryonic metabolism has been to evaluate consumption and production of several key energetic substrates (glucose, pyruvate, and lactate) using microfluorometric enzymatic assays. These assays are performed manually using constriction pipets, which greatly limits the utility of this system. Through multilayer soft-lithography, we have designed a microfluidic device that can perform these assays in an automated fashion. Following manual loading of samples and enzyme cocktail reagents, this system performs sample and enzyme cocktail aliquotting, mixing of reagents, data acquisition, and data analysis without operator intervention. Optimization of design and operating regimens has resulted in the ability to perform serial measurements of glucose, pyruvate, and lactate in triplicate with submicroliter sample volumes within 5 min. The current architecture allows for automated analysis of 10 samples and intermittent calibration over a 3 h period. Standard curves generated for each metabolite have correlation coefficients that routinely exceed 0.99. With the use of a standard epifluorescent microscope and CCD camera, linearity is obtained with metabolite concentrations in the low micromolar range (low femtomoles of total analyte). This system is inherently flexible, being easily adapted for any NAD(P)H-based assay and scaled up in terms of sample ports. Open source JAVA-based software allows for simple alterations in routine algorithms. Furthermore, this device can be used as a standalone device in which media samples are loaded or be integrated into microfluidic culture systems for in line, real time metabolic evaluation. With the improved throughput and flexibility of this system, many barriers to evaluating metabolism of embryos and single cells are eliminated. As a proof of principle, metabolic activities of single murine embryos were evaluated using this device.

Skylab S-191 spectrometer single spectral scan analysis program. [user manual

NASA Technical Reports Server (NTRS)

Downes, E. L.

1974-01-01

Documentation and user information for the S-191 single spectral scan analysis program are reported. A breakdown of the computational algorithms is supplied, followed by the program listing and examples of sample output. A copy of the flow chart which describes the driver routine in the body of the main program segment is included.

Development of Magnetic Nanomaterials and Devices for Biological Applications

DTIC Science & Technology

2007-10-30

analysis. Suitable crystals for the X-ray diffraction analysis were grown as dark red plates from a saturated hexane solution of [ Co3 (CO)9CCH3] at 4 ºC...Commercially available magnetic nanoparticles are suitable for cell separation where a large number of particles are used to separate a single cell...from a sample. The magnetic moment of these particles is not high enough to enable the separation of single antigen molecules using a single particle

Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules

PubMed Central

Panzeri, Francesco

2017-01-01

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions. PMID:28419142

Preparation and Loading Process of Single Crystalline Samples into a Gas Environmental Cell Holder for In Situ Atomic Resolution Scanning Transmission Electron Microscopic Observation.

PubMed

Straubinger, Rainer; Beyer, Andreas; Volz, Kerstin

2016-06-01

A reproducible way to transfer a single crystalline sample into a gas environmental cell holder for in situ transmission electron microscopic (TEM) analysis is shown in this study. As in situ holders have only single-tilt capability, it is necessary to prepare the sample precisely along a specific zone axis. This can be achieved by a very accurate focused ion beam lift-out preparation. We show a step-by-step procedure to prepare the sample and transfer it into the gas environmental cell. The sample material is a GaP/Ga(NAsP)/GaP multi-quantum well structure on Si. Scanning TEM observations prove that it is possible to achieve atomic resolution at very high temperatures in a nitrogen environment of 100,000 Pa.

Optofluidic Cell Selection from Complex Microbial Communities for Single-Genome Analysis

PubMed Central

Landry, Zachary C.; Giovanonni, Stephen J.; Quake, Stephen R.; Blainey, Paul C.

2013-01-01

Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations. Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences. A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity. Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy. PMID:24060116

Detailed Surface Analysis Of Incremental Centrifugal Barrel Polishing (CBP) Of Single-Crystal Niobium Samples

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

Palczewski, Ari D.; Tian, Hui; Trofimova, Olga

2011-07-01

We performed Centrifugal Barrel Polishing (CBP) on single crystal niobium samples/coupons housed in a stainless steel sample holder following the polishing recipe developed at Fermi Lab (FNAL) in 2011 \\cite{C. A. Cooper 2011}. Post CBP, the sample coupons were analyzed for surface roughness, crystal composition and structure, and particle contamination. Following the initial analysis each coupon was high pressure rinsed (HRP) and analyzed for the effectiveness of contamination removal. We were able to obtain the mirror like surface finish after the final stage of tumbling, although some defects and embedded particles remained. In addition, standard HPR appears to have littlemore » effect on removing embedded particles which remain after each tumbling step, although final polishing media removal was partially affected by standard/extended HPR.« less

U/Th dating by SHRIMP RG ion-microprobe mass spectrometry using single ion-exchange beads

NASA Astrophysics Data System (ADS)

Bischoff, James L.; Wooden, Joe; Murphy, Fred; Williams, Ross W.

2005-04-01

We present a new analytical method for U-series isotopes using the SHRIMP RG (Sensitive High mass Resolution Ion MicroProbe) mass spectrometer that utilizes the preconcentration of the U-series isotopes from a sample onto a single ion-exchange bead. Ion-microprobe mass spectrometry is capable of producing Th ionization efficiencies in excess of 2%. Analytical precision is typically better than alpha spectroscopy, but not as good as thermal ionization mass spectroscopy (TIMS) and inductively coupled plasma multicollector mass spectrometry (ICP-MS). Like TIMS and ICP-MS the method allows analysis of small samples sizes, but also adds the advantage of rapidity of analysis. A major advantage of ion-microprobe analysis is that U and Th isotopes are analyzed in the same bead, simplifying the process of chemical separation. Analytical time on the instrument is ˜60 min per sample, and a single instrument-loading can accommodate 15-20 samples to be analyzed in a 24-h day. An additional advantage is that the method allows multiple reanalyses of the same bead and that samples can be archived for reanalysis at a later time. Because the ion beam excavates a pit only a few μm deep, the mount can later be repolished and reanalyzed numerous times. The method described of preconcentrating a low concentration sample onto a small conductive substrate to allow ion-microprobe mass spectrometry is potentially applicable to many other systems.

U/Th dating by SHRIMP RG ion-microprobe mass spectrometry using single ion-exchange beads

USGS Publications Warehouse

Bischoff, J.L.; Wooden, J.; Murphy, F.; Williams, Ross W.

2005-01-01

We present a new analytical method for U-series isotopes using the SHRIMP RG (Sensitive High mass Resolution Ion MicroProbe) mass spectrometer that utilizes the preconcentration of the U-series isotopes from a sample onto a single ion-exchange bead. Ion-microprobe mass spectrometry is capable of producing Th ionization efficiencies in excess of 2%. Analytical precision is typically better than alpha spectroscopy, but not as good as thermal ionization mass spectroscopy (TIMS) and inductively coupled plasma multicollector mass spectrometry (ICP-MS). Like TIMS and ICP-MS the method allows analysis of small samples sizes, but also adds the advantage of rapidity of analysis. A major advantage of ion-microprobe analysis is that U and Th isotopes are analyzed in the same bead, simplifying the process of chemical separation. Analytical time on the instrument is ???60 min per sample, and a single instrument-loading can accommodate 15-20 samples to be analyzed in a 24-h day. An additional advantage is that the method allows multiple reanalyses of the same bead and that samples can be archived for reanalysis at a later time. Because the ion beam excavates a pit only a few ??m deep, the mount can later be repolished and reanalyzed numerous times. The method described of preconcentrating a low concentration sample onto a small conductive substrate to allow ion-microprobe mass spectrometry is potentially applicable to many other systems. Copyright ?? 2005 Elsevier Ltd.

Development of automated high throughput single molecular microfluidic detection platform for signal transduction analysis

NASA Astrophysics Data System (ADS)

Huang, Po-Jung; Baghbani Kordmahale, Sina; Chou, Chao-Kai; Yamaguchi, Hirohito; Hung, Mien-Chie; Kameoka, Jun

2016-03-01

Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. "microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)"we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.

Multiplexed droplet single-cell RNA-sequencing using natural genetic variation.

PubMed

Kang, Hyun Min; Subramaniam, Meena; Targ, Sasha; Nguyen, Michelle; Maliskova, Lenka; McCarthy, Elizabeth; Wan, Eunice; Wong, Simon; Byrnes, Lauren; Lanata, Cristina M; Gate, Rachel E; Mostafavi, Sara; Marson, Alexander; Zaitlen, Noah; Criswell, Lindsey A; Ye, Chun Jimmie

2018-01-01

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples.

Repeated stool sampling and use of multiple techniques enhance the sensitivity of helminth diagnosis: a cross-sectional survey in southern Lao People's Democratic Republic.

PubMed

Sayasone, Somphou; Utzinger, Jürg; Akkhavong, Kongsap; Odermatt, Peter

2015-01-01

Intestinal parasitic infections are common in Lao People's Democratic Republic (Lao PDR). We investigated the accuracy of the Kato-Katz (KK) technique in relation to varying stool sampling efforts, and determined the effect of the concurrent use of a quantitative formalin-ethyl acetate concentration technique (FECT) for helminth diagnosis and appraisal of concomitant infections. The study was carried out between March and May 2006 in Champasack province, southern Lao PDR. Overall, 485 individuals aged ≥6 months who provided three stool samples were included in the final analysis. All stool samples were subjected to the KK technique. Additionally, one stool sample per individual was processed by FECT. Diagnosis was done under a light microscope by experienced laboratory technicians. Analysis of three stool samples with KK plus a single FECT was considered as diagnostic 'gold' standard and resulted in prevalence estimates of hookworm, Opisthorchis viverrini, Ascaris lumbricoides, Trichuris trichiura and Schistosoma mekongi infection of 77.9%, 65.0%, 33.4%, 26.2% and 24.3%, respectively. As expected, a single KK and a single FECT missed a considerable number of infections. While our diagnostic 'gold' standard produced similar results than those obtained by a mathematical model for most helminth infections, the 'true' prevalence predicted by the model for S. mekongi (28.1%) was somewhat higher than after multiple KK plus a single FECT (24.3%). In the current setting, triplicate KK plus a single FECT diagnosed helminth infections with high sensitivity. Hence, such a diagnostic approach might be utilised for generating high-quality baseline data, assessing anthelminthic drug efficacy and rigorous monitoring of community interventions. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of single nucleotide polymorphisms in case-control studies.

PubMed

Li, Yonghong; Shiffman, Dov; Oberbauer, Rainer

2011-01-01

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variants in the human genome. SNPs are known to modify susceptibility to complex diseases. We describe and discuss methods used to identify SNPs associated with disease in case-control studies. An outline on study population selection, sample collection and genotyping platforms is presented, complemented by SNP selection, data preprocessing and analysis.

Nanoarchaeota, Their Sulfolobales Host, and Nanoarchaeota Virus Distribution across Yellowstone National Park Hot Springs

PubMed Central

Munson-McGee, Jacob H.; Field, Erin K.; Bateson, Mary; Rooney, Colleen; Stepanauskas, Ramunas

2015-01-01

Nanoarchaeota are obligate symbionts with reduced genomes first described from marine thermal vent environments. Here, both community metagenomics and single-cell analysis revealed the presence of Nanoarchaeota in high-temperature (∼90°C), acidic (pH ≈ 2.5 to 3.0) hot springs in Yellowstone National Park (YNP) (United States). Single-cell genome analysis of two cells resulted in two nearly identical genomes, with an estimated full length of 650 kbp. Genome comparison showed that these two cells are more closely related to the recently proposed Nanobsidianus stetteri from a more neutral YNP hot spring than to the marine Nanoarchaeum equitans. Single-cell and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis of environmental hot spring samples identified the host of the YNP Nanoarchaeota as a Sulfolobales species known to inhabit the hot springs. Furthermore, we demonstrate that Nanoarchaeota are widespread in acidic to near neutral hot springs in YNP. An integrated viral sequence was also found within one Nanoarchaeota single-cell genome and further analysis of the purified viral fraction from environmental samples indicates that this is likely a virus replicating within the YNP Nanoarchaeota. PMID:26341207

Inorganic, Radioisotopic, and Organic Analysis of 241-AP-101 Tank Waste

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

Fiskum, S.K.; Bredt, P.R.; Campbell, J.A.

2000-10-17

Battelle received five samples from Hanford waste tank 241-AP-101, taken at five different depths within the tank. No visible solids or organic layer were observed in the individual samples. Individual sample densities were measured, then the five samples were mixed together to provide a single composite. The composite was homogenized and representative sub-samples taken for inorganic, radioisotopic, and organic analysis. All analyses were performed on triplicate sub-samples of the composite material. The sample composite did not contain visible solids or an organic layer. A subsample held at 10 C for seven days formed no visible solids.

Toward single-cell analysis by plume collimation in laser ablation electrospray ionization mass spectrometry.

PubMed

Stolee, Jessica A; Vertes, Akos

2013-04-02

Ambient ionization methods for mass spectrometry have enabled the in situ and in vivo analysis of biological tissues and cells. When an etched optical fiber is used to deliver laser energy to a sample in laser ablation electrospray ionization (LAESI) mass spectrometry, the analysis of large single cells becomes possible. However, because in this arrangement the ablation plume expands in three dimensions, only a small portion of it is ionized by the electrospray. Here we show that sample ablation within a capillary helps to confine the radial expansion of the plume. Plume collimation, due to the altered expansion dynamics, leads to greater interaction with the electrospray plume resulting in increased ionization efficiency, reduced limit of detection (by a factor of ~13, reaching 600 amol for verapamil), and extended dynamic range (6 orders of magnitude) compared to conventional LAESI. This enhanced sensitivity enables the analysis of a range of metabolites from small cell populations and single cells in the ambient environment. This technique has the potential to be integrated with flow cytometry for high-throughput metabolite analysis of sorted cells.

Relative effectiveness of kinetic analysis vs single point readings for classifying environmental samples based on community-level physiological profiles (CLPP)

NASA Technical Reports Server (NTRS)

Garland, J. L.; Mills, A. L.; Young, J. S.

2001-01-01

The relative effectiveness of average-well-color-development-normalized single-point absorbance readings (AWCD) vs the kinetic parameters mu(m), lambda, A, and integral (AREA) of the modified Gompertz equation fit to the color development curve resulting from reduction of a redox sensitive dye from microbial respiration of 95 separate sole carbon sources in microplate wells was compared for a dilution series of rhizosphere samples from hydroponically grown wheat and potato ranging in inoculum densities of 1 x 10(4)-4 x 10(6) cells ml-1. Patterns generated with each parameter were analyzed using principal component analysis (PCA) and discriminant function analysis (DFA) to test relative resolving power. Samples of equivalent cell density (undiluted samples) were correctly classified by rhizosphere type for all parameters based on DFA analysis of the first five PC scores. Analysis of undiluted and 1:4 diluted samples resulted in misclassification of at least two of the wheat samples for all parameters except the AWCD normalized (0.50 abs. units) data, and analysis of undiluted, 1:4, and 1:16 diluted samples resulted in misclassification for all parameter types. Ordination of samples along the first principal component (PC) was correlated to inoculum density in analyses performed on all of the kinetic parameters, but no such influence was seen for AWCD-derived results. The carbon sources responsible for classification differed among the variable types with the exception of AREA and A, which were strongly correlated. These results indicate that the use of kinetic parameters for pattern analysis in CLPP may provide some additional information, but only if the influence of inoculum density is carefully considered. c2001 Elsevier Science Ltd. All rights reserved.

Scalable multi-sample single-cell data analysis by Partition-Assisted Clustering and Multiple Alignments of Networks

PubMed Central

Samusik, Nikolay; Wang, Xiaowei; Guan, Leying; Nolan, Garry P.

2017-01-01

Mass cytometry (CyTOF) has greatly expanded the capability of cytometry. It is now easy to generate multiple CyTOF samples in a single study, with each sample containing single-cell measurement on 50 markers for more than hundreds of thousands of cells. Current methods do not adequately address the issues concerning combining multiple samples for subpopulation discovery, and these issues can be quickly and dramatically amplified with increasing number of samples. To overcome this limitation, we developed Partition-Assisted Clustering and Multiple Alignments of Networks (PAC-MAN) for the fast automatic identification of cell populations in CyTOF data closely matching that of expert manual-discovery, and for alignments between subpopulations across samples to define dataset-level cellular states. PAC-MAN is computationally efficient, allowing the management of very large CyTOF datasets, which are increasingly common in clinical studies and cancer studies that monitor various tissue samples for each subject. PMID:29281633

FANTOM5 CAGE profiles of human and mouse samples.

PubMed

Noguchi, Shuhei; Arakawa, Takahiro; Fukuda, Shiro; Furuno, Masaaki; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Kaida, Kaoru; Kaiho, Ai; Kanamori-Katayama, Mutsumi; Kawashima, Tsugumi; Kojima, Miki; Kubosaki, Atsutaka; Manabe, Ri-Ichiroh; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakazato, Kenichi; Ninomiya, Noriko; Nishiyori-Sueki, Hiromi; Noma, Shohei; Saijyo, Eri; Saka, Akiko; Sakai, Mizuho; Simon, Christophe; Suzuki, Naoko; Tagami, Michihira; Watanabe, Shoko; Yoshida, Shigehiro; Arner, Peter; Axton, Richard A; Babina, Magda; Baillie, J Kenneth; Barnett, Timothy C; Beckhouse, Anthony G; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Carlisle, Ailsa J; Clevers, Hans C; Davis, Carrie A; Detmar, Michael; Dohi, Taeko; Edge, Albert S B; Edinger, Matthias; Ehrlund, Anna; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Farach-Carson, Mary C; Faulkner, Geoffrey J; Ferrai, Carmelo; Fisher, Malcolm E; Forrester, Lesley M; Fujita, Rie; Furusawa, Jun-Ichi; Geijtenbeek, Teunis B; Gingeras, Thomas; Goldowitz, Daniel; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J; Hamaguchi, Masahide; Hara, Mitsuko; Hasegawa, Yuki; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J; Hume, David A; Ikawa, Tomokatsu; Ishizu, Yuri; Kai, Chieko; Kawamoto, Hiroshi; Kawamura, Yuki I; Kempfle, Judith S; Kenna, Tony J; Kere, Juha; Khachigian, Levon M; Kitamura, Toshio; Klein, Sarah; Klinken, S Peter; Knox, Alan J; Kojima, Soichi; Koseki, Haruhiko; Koyasu, Shigeo; Lee, Weonju; Lennartsson, Andreas; Mackay-Sim, Alan; Mejhert, Niklas; Mizuno, Yosuke; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Morris, Kelly J; Motohashi, Hozumi; Mummery, Christine L; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Pradhan-Bhatt, Swati; Qin, Xian-Yang; Rehli, Michael; Rizzu, Patrizia; Roy, Sugata; Sajantila, Antti; Sakaguchi, Shimon; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schmidl, Christian; Schneider, Claudio; Schulze-Tanzil, Gundula G; Schwegmann, Anita; Sheng, Guojun; Shin, Jay W; Sugiyama, Daisuke; Sugiyama, Takaaki; Summers, Kim M; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tomoiu, Andru; Toyoda, Hiroo; van de Wetering, Marc; van den Berg, Linda M; Verardo, Roberto; Vijayan, Dipti; Wells, Christine A; Winteringham, Louise N; Wolvetang, Ernst; Yamaguchi, Yoko; Yamamoto, Masayuki; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Yonekura, Yohei; Zhang, Peter G; Zucchelli, Silvia; Abugessaisa, Imad; Arner, Erik; Harshbarger, Jayson; Kondo, Atsushi; Lassmann, Timo; Lizio, Marina; Sahin, Serkan; Sengstag, Thierry; Severin, Jessica; Shimoji, Hisashi; Suzuki, Masanori; Suzuki, Harukazu; Kawai, Jun; Kondo, Naoto; Itoh, Masayoshi; Daub, Carsten O; Kasukawa, Takeya; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide

2017-08-29

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

FANTOM5 CAGE profiles of human and mouse samples

PubMed Central

Noguchi, Shuhei; Arakawa, Takahiro; Fukuda, Shiro; Furuno, Masaaki; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Kaida, Kaoru; Kaiho, Ai; Kanamori-Katayama, Mutsumi; Kawashima, Tsugumi; Kojima, Miki; Kubosaki, Atsutaka; Manabe, Ri-ichiroh; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakazato, Kenichi; Ninomiya, Noriko; Nishiyori-Sueki, Hiromi; Noma, Shohei; Saijyo, Eri; Saka, Akiko; Sakai, Mizuho; Simon, Christophe; Suzuki, Naoko; Tagami, Michihira; Watanabe, Shoko; Yoshida, Shigehiro; Arner, Peter; Axton, Richard A.; Babina, Magda; Baillie, J. Kenneth; Barnett, Timothy C.; Beckhouse, Anthony G.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Carlisle, Ailsa J.; Clevers, Hans C.; Davis, Carrie A.; Detmar, Michael; Dohi, Taeko; Edge, Albert S.B.; Edinger, Matthias; Ehrlund, Anna; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Farach-Carson, Mary C.; Faulkner, Geoffrey J.; Ferrai, Carmelo; Fisher, Malcolm E.; Forrester, Lesley M.; Fujita, Rie; Furusawa, Jun-ichi; Geijtenbeek, Teunis B.; Gingeras, Thomas; Goldowitz, Daniel; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J.; Hamaguchi, Masahide; Hara, Mitsuko; Hasegawa, Yuki; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J.; Hume, David A.; Ikawa, Tomokatsu; Ishizu, Yuri; Kai, Chieko; Kawamoto, Hiroshi; Kawamura, Yuki I.; Kempfle, Judith S.; Kenna, Tony J.; Kere, Juha; Khachigian, Levon M.; Kitamura, Toshio; Klein, Sarah; Klinken, S. Peter; Knox, Alan J.; Kojima, Soichi; Koseki, Haruhiko; Koyasu, Shigeo; Lee, Weonju; Lennartsson, Andreas; Mackay-sim, Alan; Mejhert, Niklas; Mizuno, Yosuke; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Morris, Kelly J.; Motohashi, Hozumi; Mummery, Christine L.; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A.; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Pradhan-Bhatt, Swati; Qin, Xian-Yang; Rehli, Michael; Rizzu, Patrizia; Roy, Sugata; Sajantila, Antti; Sakaguchi, Shimon; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schmidl, Christian; Schneider, Claudio; Schulze-Tanzil, Gundula G.; Schwegmann, Anita; Sheng, Guojun; Shin, Jay W.; Sugiyama, Daisuke; Sugiyama, Takaaki; Summers, Kim M.; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tomoiu, Andru; Toyoda, Hiroo; van de Wetering, Marc; van den Berg, Linda M.; Verardo, Roberto; Vijayan, Dipti; Wells, Christine A.; Winteringham, Louise N.; Wolvetang, Ernst; Yamaguchi, Yoko; Yamamoto, Masayuki; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Yonekura, Yohei; Zhang, Peter G.; Zucchelli, Silvia; Abugessaisa, Imad; Arner, Erik; Harshbarger, Jayson; Kondo, Atsushi; Lassmann, Timo; Lizio, Marina; Sahin, Serkan; Sengstag, Thierry; Severin, Jessica; Shimoji, Hisashi; Suzuki, Masanori; Suzuki, Harukazu; Kawai, Jun; Kondo, Naoto; Itoh, Masayoshi; Daub, Carsten O.; Kasukawa, Takeya; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R.R.; Hayashizaki, Yoshihide

2017-01-01

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities. PMID:28850106

Quantitative x-ray phase-contrast imaging using a single grating of comparable pitch to sample feature size.

PubMed

Morgan, Kaye S; Paganin, David M; Siu, Karen K W

2011-01-01

The ability to quantitatively retrieve transverse phase maps during imaging by using coherent x rays often requires a precise grating or analyzer-crystal-based setup. Imaging of live animals presents further challenges when these methods require multiple exposures for image reconstruction. We present a simple method of single-exposure, single-grating quantitative phase contrast for a regime in which the grating period is much greater than the effective pixel size. A grating is used to create a high-visibility reference pattern incident on the sample, which is distorted according to the complex refractive index and thickness of the sample. The resolution, along a line parallel to the grating, is not restricted by the grating spacing, and the detector resolution becomes the primary determinant of the spatial resolution. We present a method of analysis that maps the displacement of interrogation windows in order to retrieve a quantitative phase map. Application of this analysis to the imaging of known phantoms shows excellent correspondence.

Single-molecule fluorescence microscopy review: shedding new light on old problems

PubMed Central

Shashkova, Sviatlana

2017-01-01

Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called ‘green revolution’, has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. PMID:28694303

7 CFR 160.202 - Fees generally for laboratory analysis and testing.

Code of Federal Regulations, 2012 CFR

2012-01-01

... turpentine. (See Note 3). (1) Comprehensive analysis to determine purity, specification compliance, or other... related to quality of utility. (i) Single Sample: (A) Rosin—$14.00. (B) Turpentine—$10.00. (ii) Two or more samples tested at same time: (A) Rosin—per sample—$10.00. (B) Turpentine—per sample—$8.00. Note 3...

7 CFR 160.202 - Fees generally for laboratory analysis and testing.

Code of Federal Regulations, 2011 CFR

2011-01-01

... turpentine. (See Note 3). (1) Comprehensive analysis to determine purity, specification compliance, or other... related to quality of utility. (i) Single Sample: (A) Rosin—$14.00. (B) Turpentine—$10.00. (ii) Two or more samples tested at same time: (A) Rosin—per sample—$10.00. (B) Turpentine—per sample—$8.00. Note 3...

7 CFR 160.202 - Fees generally for laboratory analysis and testing.

Code of Federal Regulations, 2013 CFR

2013-01-01

... turpentine. (See Note 3). (1) Comprehensive analysis to determine purity, specification compliance, or other... related to quality of utility. (i) Single Sample: (A) Rosin—$14.00. (B) Turpentine—$10.00. (ii) Two or more samples tested at same time: (A) Rosin—per sample—$10.00. (B) Turpentine—per sample—$8.00. Note 3...

7 CFR 160.202 - Fees generally for laboratory analysis and testing.

Code of Federal Regulations, 2010 CFR

2010-01-01

... turpentine. (See Note 3). (1) Comprehensive analysis to determine purity, specification compliance, or other... related to quality of utility. (i) Single Sample: (A) Rosin—$14.00. (B) Turpentine—$10.00. (ii) Two or more samples tested at same time: (A) Rosin—per sample—$10.00. (B) Turpentine—per sample—$8.00. Note 3...

7 CFR 160.202 - Fees generally for laboratory analysis and testing.

Code of Federal Regulations, 2014 CFR

2014-01-01

... turpentine. (See Note 3). (1) Comprehensive analysis to determine purity, specification compliance, or other... related to quality of utility. (i) Single Sample: (A) Rosin—$14.00. (B) Turpentine—$10.00. (ii) Two or more samples tested at same time: (A) Rosin—per sample—$10.00. (B) Turpentine—per sample—$8.00. Note 3...

Exploratory Two-Level Analysis of Individual- and School-Level Factors on Truant Youth Emotional/Psychological Functioning

ERIC Educational Resources Information Center

Dembo, Richard; Wareham, Jennifer; Schmeidler, James; Winters, Ken C.

2016-01-01

Research on samples of truant adolescents is limited, with little known about mental health problems among truant youths. This study provided an exploratory, multilevel examination of mental health problems for a sample of 300 truant adolescents. Confirmatory factor analysis indicated a single factor of multiple mental health problems at the…

Bias and Precision of Measures of Association for a Fixed-Effect Multivariate Analysis of Variance Model

ERIC Educational Resources Information Center

Kim, Soyoung; Olejnik, Stephen

2005-01-01

The sampling distributions of five popular measures of association with and without two bias adjusting methods were examined for the single factor fixed-effects multivariate analysis of variance model. The number of groups, sample sizes, number of outcomes, and the strength of association were manipulated. The results indicate that all five…

An Applied Example of Pooled Time Series Analysis: Cardiovascular Reactivity to Stressors in Children with Autism

ERIC Educational Resources Information Center

Hoeppner, Bettina B.; Goodwin, Matthew S.; Velicer, Wayne F.; Heltshe, James

2007-01-01

The advent of telemetric devices that sample data extensively over time has facilitated single subject or idiographic research to intensively study a single person over time. One of the challenges of idiographic research is combining single subject results to determine generalizability across subjects. This article demonstrates the first…

Indel analysis by droplet digital PCR: a sensitive method for DNA mixture detection and chimerism analysis.

PubMed

Santurtún, Ana; Riancho, José A; Arozamena, Jana; López-Duarte, Mónica; Zarrabeitia, María T

2017-01-01

Several methods have been developed to determinate genetic profiles from a mixed samples and chimerism analysis in transplanted patients. The aim of this study was to explore the effectiveness of using the droplet digital PCR (ddPCR) for mixed chimerism detection (a mixture of genetic profiles resulting after allogeneic hematopoietic stem cell transplantation (HSCT)). We analyzed 25 DNA samples from patients who had undergone HSCT and compared the performance of ddPCR and two established methods for chimerism detection, based upon the Indel and STRs analysis, respectively. Additionally, eight artificial mixture DNA samples were created to evaluate the sensibility of ddPCR. Our results show that the chimerism percentages estimated by the analysis of a single Indel using ddPCR were very similar to those calculated by the amplification of 15 STRs (r 2  = 0.970) and with the results obtained by the amplification of 38 Indels (r 2  = 0.975). Moreover, the amplification of a single Indel by ddPCR was sensitive enough to detect a minor DNA contributor comprising down to 0.5 % of the sample. We conclude that ddPCR can be a powerful tool for the determination of a genetic profile of forensic mixtures and clinical chimerism analysis when traditional techniques are not sensitive enough.

Sequence analysis of cultivated strawberry (Fragaria × ananassa Duch.) using microdissected single somatic chromosomes.

PubMed

Yanagi, Tomohiro; Shirasawa, Kenta; Terachi, Mayuko; Isobe, Sachiko

2017-01-01

Cultivated strawberry ( Fragaria  ×  ananassa Duch.) has homoeologous chromosomes because of allo-octoploidy. For example, two homoeologous chromosomes that belong to different sub-genome of allopolyploids have similar base sequences. Thus, when conducting de novo assembly of DNA sequences, it is difficult to determine whether these sequences are derived from the same chromosome. To avoid the difficulties associated with homoeologous chromosomes and demonstrate the possibility of sequencing allopolyploids using single chromosomes, we conducted sequence analysis using microdissected single somatic chromosomes of cultivated strawberry. Three hundred and ten somatic chromosomes of the Japanese octoploid strawberry 'Reiko' were individually selected under a light microscope using a microdissection system. DNA from 288 of the dissected chromosomes was successfully amplified using a DNA amplification kit. Using next-generation sequencing, we decoded the base sequences of the amplified DNA segments, and on the basis of mapping, we identified DNA sequences from 144 samples that were best matched to the reference genomes of the octoploid strawberry, F.  ×  ananassa , and the diploid strawberry, F. vesca . The 144 samples were classified into seven pseudo-molecules of F. vesca . The coverage rates of the DNA sequences from the single chromosome onto all pseudo-molecular sequences varied from 3 to 29.9%. We demonstrated an efficient method for sequence analysis of allopolyploid plants using microdissected single chromosomes. On the basis of our results, we believe that whole-genome analysis of allopolyploid plants can be enhanced using methodology that employs microdissected single chromosomes.

Tandem array of nanoelectronic readers embedded coplanar to a fluidic nanochannel for correlated single biopolymer analysis

PubMed Central

Lesser-Rojas, Leonardo; Sriram, K. K.; Liao, Kuo-Tang; Lai, Shui-Chin; Kuo, Pai-Chia; Chu, Ming-Lee; Chou, Chia-Fu

2014-01-01

We have developed a two-step electron-beam lithography process to fabricate a tandem array of three pairs of tip-like gold nanoelectronic detectors with electrode gap size as small as 9 nm, embedded in a coplanar fashion to 60 nm deep, 100 nm wide, and up to 150 μm long nanochannels coupled to a world-micro-nanofluidic interface for easy sample introduction. Experimental tests with a sealed device using DNA-protein complexes demonstrate the coplanarity of the nanoelectrodes to the nanochannel surface. Further, this device could improve transverse current detection by correlated time-of-flight measurements of translocating samples, and serve as an autocalibrated velocimeter and nanoscale tandem Coulter counters for single molecule analysis of heterogeneous samples. PMID:24753731

Pore structure of raw and purified HiPco single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Cinke, Martin; Li, Jing; Chen, Bin; Cassell, Alan; Delzeit, Lance; Han, Jie; Meyyappan, M.

2002-10-01

Very high purity single-walled carbon nanotubes (SWNTs) were obtained from HiPco SWNT samples containing Fe particles by a two-step purification process. The raw and purified samples were characterized using high resolution transmission electron microscopy (HRTEM), Raman spectroscopy and thermogravimetric analysis (TGA). The purified sample consists of ˜0.4% Fe and the process does not seem to introduce any additional defects. The N 2 adsorption isotherm studies at 77 K reveal that the total surface area of the purified sample increases to 1587 m 2/g from 567 m 2/g for the raw material, which is the highest value reported for SWNTs.

Single Crystal Synthesis and STM Studies of High Temperature Superconductors

NASA Technical Reports Server (NTRS)

Barrientos, Alfonso

1997-01-01

This is a final report for the work initiated in September of 1994 under the grant NAG8-1085 - NASA/OMU, on the fabrication of bulk and single crystal synthesis, specific heat measuring and STM studies of high temperature superconductors. Efforts were made to fabricate bulk and single crystals of mercury based superconducting material. A systematic thermal analysis on the precursors for the corresponding oxides and carbonates were carried out to synthesized bulk samples. Bulk material was used as seed in an attempt to grow single crystals by a two-step self flux process. On the other hand bulk samples were characterized by x-ray diffraction, electrical resistivity and magnetic susceptibility, We studied the specific heat behavior in the range from 80 to 300 K. Some preliminary attempts were made to study the atomic morphology of our samples. As part of our efforts we built an ac susceptibility apparatus for measuring the transition temperature of our sintered samples.

In Vitro Evaluation and Mechanism Analysis of the Fiber Shedding Property of Textile Pile Debridement Materials

PubMed Central

Fu, Yijun; Xie, Qixue; Lao, Jihong; Wang, Lu

2016-01-01

Fiber shedding is a critical problem in biomedical textile debridement materials, which leads to infection and impairs wound healing. In this work, single fiber pull-out test was proposed as an in vitro evaluation for the fiber shedding property of a textile pile debridement material. Samples with different structural design (pile densities, numbers of ground yarns and coating times) were prepared and estimated under this testing method. Results show that single fiber pull-out test offers an appropriate in vitro evaluation for the fiber shedding property of textile pile debridement materials. Pull-out force for samples without back-coating exhibited a slight escalating trend with the supplement in pile density and number of ground yarn plies, while back-coating process significantly raised the single fiber pull-out force. For fiber shedding mechanism analysis, typical pull-out behavior and failure modes of the single fiber pull-out test were analyzed in detail. Three failure modes were found in this study, i.e., fiber slippage, coating point rupture and fiber breakage. In summary, to obtain samples with desirable fiber shedding property, fabric structural design, preparation process and raw materials selection should be taken into full consideration. PMID:28773428

Spectrochemical analysis of powdered biological samples using transversely excited atmospheric carbon dioxide laser plasma excitation

NASA Astrophysics Data System (ADS)

Zivkovic, Sanja; Momcilovic, Milos; Staicu, Angela; Mutic, Jelena; Trtica, Milan; Savovic, Jelena

2017-02-01

The aim of this study was to develop a simple laser induced breakdown spectroscopy (LIBS) method for quantitative elemental analysis of powdered biological materials based on laboratory prepared calibration samples. The analysis was done using ungated single pulse LIBS in ambient air at atmospheric pressure. Transversely-Excited Atmospheric pressure (TEA) CO2 laser was used as an energy source for plasma generation on samples. The material used for the analysis was a blue-green alga Spirulina, widely used in food and pharmaceutical industries and also in a few biotechnological applications. To demonstrate the analytical potential of this particular LIBS system the obtained spectra were compared to the spectra obtained using a commercial LIBS system based on pulsed Nd:YAG laser. A single sample of known concentration was used to estimate detection limits for Ba, Ca, Fe, Mg, Mn, Si and Sr and compare detection power of these two LIBS systems. TEA CO2 laser based LIBS was also applied for quantitative analysis of the elements in powder Spirulina samples. Analytical curves for Ba, Fe, Mg, Mn and Sr were constructed using laboratory produced matrix-matched calibration samples. Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used as the reference technique for elemental quantification, and reasonably well agreement between ICP and LIBS data was obtained. Results confirm that, in respect to its sensitivity and precision, TEA CO2 laser based LIBS can be successfully applied for quantitative analysis of macro and micro-elements in algal samples. The fact that nearly all classes of materials can be prepared as powders implies that the proposed method could be easily extended to a quantitative analysis of different kinds of materials, organic, biological or inorganic.

Compartmental genomics in living cells revealed by single-cell nanobiopsy.

PubMed

Actis, Paolo; Maalouf, Michelle M; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R Adam; Pourmand, Nader

2014-01-28

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

Palladium-based Mass-Tag Cell Barcoding with a Doublet-Filtering Scheme and Single Cell Deconvolution Algorithm

PubMed Central

Zunder, Eli R.; Finck, Rachel; Behbehani, Gregory K.; Amir, El-ad D.; Krishnaswamy, Smita; Gonzalez, Veronica D.; Lorang, Cynthia G.; Bjornson, Zach; Spitzer, Matthew H.; Bodenmiller, Bernd; Fantl, Wendy J.; Pe’er, Dana; Nolan, Garry P.

2015-01-01

SUMMARY Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption, and shortens instrument measurement time. Here, we present an optimized MCB protocol with several improvements over previously described methods. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3–4 h, not including sample acquisition time of ~1 h per million cells. PMID:25612231

Fourier crosstalk analysis of multislice and cone-beam helical CT

NASA Astrophysics Data System (ADS)

La Riviere, Patrick J.

2004-05-01

Multi-slice helical CT scanners allow for much faster scanning and better x-ray utilization than do their single-slice predecessors, but they engender considerably more complicated data sampling patterns due to the interlacing of the samples from different rows as the patient is translated. Characterizing and optimizing this sampling is challenging because the conebeam geometry of such scanners means that the projections measured by each detector row are at least slightly oblique, making it difficult to apply standard multidimensional sampling analyses. In this study, we seek to apply a more general framework for analyzing sampled imaging systems known as Fourier crosstalk analysis. Our purpose in this preliminary work is to compare the information content of the data acquired in three different scanner geometries and operating conditions with ostensibly equivalent volume coverage and average longitudinal sampling interval: a single-slice scanner operating at pitch 1, a four-slice scanner operating at pitch 3 and a 15-slice scanner operating at pitch 15. We find that moving from a single-slice to a multi-slice geometry introduces longitudinal crosstalk characteristic of the longitudinal sampling interval between periods of individual each detector row, and not of the overall interlaced sampling pattern. This is attributed to data inconsistencies caused by the obliqueness of the projections in a multi-slice/conebeam configuration. However, these preliminary results suggest that the significance of this additional crosstalk actually decreases as the number of detector rows increases.

Hotspot and sampling analysis for effective maintenance and performance monitoring.

DOT National Transportation Integrated Search

2017-05-01

In this project, we propose two sampling methods addressing how much and where the agencies need to collect infrastraucture condition data for accurate Level-of-Maintenance (LOM) estimation in maintenance network with single type or multiple ty...

A generic, cost-effective, and scalable cell lineage analysis platform

PubMed Central

Biezuner, Tamir; Spiro, Adam; Raz, Ofir; Amir, Shiran; Milo, Lilach; Adar, Rivka; Chapal-Ilani, Noa; Berman, Veronika; Fried, Yael; Ainbinder, Elena; Cohen, Galit; Barr, Haim M.; Halaban, Ruth; Shapiro, Ehud

2016-01-01

Advances in single-cell genomics enable commensurate improvements in methods for uncovering lineage relations among individual cells. Current sequencing-based methods for cell lineage analysis depend on low-resolution bulk analysis or rely on extensive single-cell sequencing, which is not scalable and could be biased by functional dependencies. Here we show an integrated biochemical-computational platform for generic single-cell lineage analysis that is retrospective, cost-effective, and scalable. It consists of a biochemical-computational pipeline that inputs individual cells, produces targeted single-cell sequencing data, and uses it to generate a lineage tree of the input cells. We validated the platform by applying it to cells sampled from an ex vivo grown tree and analyzed its feasibility landscape by computer simulations. We conclude that the platform may serve as a generic tool for lineage analysis and thus pave the way toward large-scale human cell lineage discovery. PMID:27558250

Integrated single-cell genetic and transcriptional analysis suggests novel drivers of chronic lymphocytic leukemia

PubMed Central

Wang, Lili; Fan, Jean; Francis, Joshua M.; Georghiou, George; Hergert, Sarah; Li, Shuqiang; Gambe, Rutendo; Zhou, Chensheng W.; Yang, Chunxiao; Xiao, Sheng; Cin, Paola Dal; Bowden, Michaela; Kotliar, Dylan; Shukla, Sachet A.; Brown, Jennifer R.; Neuberg, Donna; Alessi, Dario R.; Zhang, Cheng-Zhong; Kharchenko, Peter V.; Livak, Kenneth J.; Wu, Catherine J.

2017-01-01

Intra-tumoral genetic heterogeneity has been characterized across cancers by genome sequencing of bulk tumors, including chronic lymphocytic leukemia (CLL). In order to more accurately identify subclones, define phylogenetic relationships, and probe genotype–phenotype relationships, we developed methods for targeted mutation detection in DNA and RNA isolated from thousands of single cells from five CLL samples. By clearly resolving phylogenic relationships, we uncovered mutated LCP1 and WNK1 as novel CLL drivers, supported by functional evidence demonstrating their impact on CLL pathways. Integrative analysis of somatic mutations with transcriptional states prompts the idea that convergent evolution generates phenotypically similar cells in distinct genetic branches, thus creating a cohesive expression profile in each CLL sample despite the presence of genetic heterogeneity. Our study highlights the potential for single-cell RNA-based targeted analysis to sensitively determine transcriptional and mutational profiles of individual cancer cells, leading to increased understanding of driving events in malignancy. PMID:28679620

Solid phase excitation-emission fluorescence method for the classification of complex substances: Cortex Phellodendri and other traditional Chinese medicines as examples.

PubMed

Gu, Yao; Ni, Yongnian; Kokot, Serge

2012-09-13

A novel, simple and direct fluorescence method for analysis of complex substances and their potential substitutes has been researched and developed. Measurements involved excitation and emission (EEM) fluorescence spectra of powdered, complex, medicinal herbs, Cortex Phellodendri Chinensis (CPC) and the similar Cortex Phellodendri Amurensis (CPA); these substances were compared and discriminated from each other and the potentially adulterated samples (Caulis mahoniae (CM) and David poplar bark (DPB)). Different chemometrics methods were applied for resolution of the complex spectra, and the excitation spectra were found to be the most informative; only the rank-ordering PROMETHEE method was able to classify the samples with single ingredients (CPA, CPC, CM) or those with binary mixtures (CPA/CPC, CPA/CM, CPC/CM). Interestingly, it was essential to use the geometrical analysis for interactive aid (GAIA) display for a full understanding of the classification results. However, these two methods, like the other chemometrics models, were unable to classify composite spectral matrices consisting of data from samples of single ingredients and binary mixtures; this suggested that the excitation spectra of the different samples were very similar. However, the method is useful for classification of single-ingredient samples and, separately, their binary mixtures; it may also be applied for similar classification work with other complex substances.

Hydrodynamic lift for single cell manipulation in a femtosecond laser fabricated optofluidic chip

NASA Astrophysics Data System (ADS)

Bragheri, Francesca; Osellame, Roberto

2017-08-01

Single cell sorting based either on fluorescence or on mechanical properties has been exploited in the last years in microfluidic devices. Hydrodynamic focusing allows increasing the efficiency of theses devices by improving the matching between the region of optical analysis and that of cell flow. Here we present a very simple solution fabricated by femtosecond laser micromachining that exploits flow laminarity in microfluidic channels to easily lift the sample flowing position to the channel portion illuminated by the optical waveguides used for single cell trapping and analysis.

Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

PubMed

Ullal, Adeeti V; Weissleder, Ralph

2015-01-01

We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization.

PubMed

Ptolemy, Adam S; Tran, Lara; Britz-McKibbin, Philip

2006-07-15

Capillary electrophoresis (CE) represents a versatile platform for integrating sample pretreatment with chemical analysis because of its ability to tune analyte electromigration and band dispersion properties in discontinuous electrolyte systems. In this article, a single-step method that combines on-line sample preconcentration with in-capillary chemical derivatization is developed for rapid, sensitive, and enantioselective analysis of micromolar levels of amino acids that lack intrinsic chromophores by CE with UV detection. Time-resolved electrophoretic studies revealed two distinct stages of amino acid band narrowing within the original long sample injection plug occurring both prior to and after in-capillary labeling via zone passing by ortho-phthalaldehyde/N-acetyl l-cysteine (OPA/NAC). This technique enabled direct analysis of d-amino acids in a 95% enantiomeric excess mixture with sub-micromolar detection limits and minimal sample handling, where the capillary functions as a preconcentrator, microreactor, and chiral selector. On-line sample preconcentration with chemical derivatization CE (SPCD-CE) was applied to study the enantioselective amino acid flux in Escherichia coli bacteria cultures, which demonstrated a unique l-Ala efflux into the extracellular medium. New strategies for high-throughput analyses of low-abundance metabolites are important for understanding fundamental physiological processes in bacteria required for screening the efficacy of new classes of antibiotics as well as altered metabolism in genetically modified mutant strains.

Accurate determination of genetic identity for a single cacao bean, using molecular markers with a nanofluidic system, ensures cocoa authentication.

PubMed

Fang, Wanping; Meinhardt, Lyndel W; Mischke, Sue; Bellato, Cláudia M; Motilal, Lambert; Zhang, Dapeng

2014-01-15

Cacao (Theobroma cacao L.), the source of cocoa, is an economically important tropical crop. One problem with the premium cacao market is contamination with off-types adulterating raw premium material. Accurate determination of the genetic identity of single cacao beans is essential for ensuring cocoa authentication. Using nanofluidic single nucleotide polymorphism (SNP) genotyping with 48 SNP markers, we generated SNP fingerprints for small quantities of DNA extracted from the seed coat of single cacao beans. On the basis of the SNP profiles, we identified an assumed adulterant variety, which was unambiguously distinguished from the authentic beans by multilocus matching. Assignment tests based on both Bayesian clustering analysis and allele frequency clearly separated all 30 authentic samples from the non-authentic samples. Distance-based principle coordinate analysis further supported these results. The nanofluidic SNP protocol, together with forensic statistical tools, is sufficiently robust to establish authentication and to verify gourmet cacao varieties. This method shows significant potential for practical application.

High Resolution Tissue Imaging Using the Single-probe Mass Spectrometry under Ambient Conditions

NASA Astrophysics Data System (ADS)

Rao, Wei; Pan, Ning; Yang, Zhibo

2015-06-01

Ambient mass spectrometry imaging (MSI) is an emerging field with great potential for the detailed spatial analysis of biological samples with minimal pretreatment. We have developed a miniaturized sampling and ionization device, the Single-probe, which uses in-situ surface micro-extraction to achieve high detection sensitivity and spatial resolution during MSI experiments. The Single-probe was coupled to a Thermo LTQ Orbitrap XL mass spectrometer and was able to create high spatial and high mass resolution MS images at 8 ± 2 and 8.5 μm on flat polycarbonate microscope slides and mouse kidney sections, respectively, which are among the highest resolutions available for ambient MSI techniques. Our proof-of-principle experiments indicate that the Single-probe MSI technique has the potential to obtain ambient MS images with very high spatial resolutions with minimal sample preparation, which opens the possibility for subcellular ambient tissue MSI to be performed in the future.

A hybrid approach to device integration on a genetic analysis platform

NASA Astrophysics Data System (ADS)

Brennan, Des; Jary, Dorothee; Kurg, Ants; Berik, Evgeny; Justice, John; Aherne, Margaret; Macek, Milan; Galvin, Paul

2012-10-01

Point-of-care (POC) systems require significant component integration to implement biochemical protocols associated with molecular diagnostic assays. Hybrid platforms where discrete components are combined in a single platform are a suitable approach to integration, where combining multiple device fabrication steps on a single substrate is not possible due to incompatible or costly fabrication steps. We integrate three devices each with a specific system functionality: (i) a silicon electro-wetting-on-dielectric (EWOD) device to move and mix sample and reagent droplets in an oil phase, (ii) a polymer microfluidic chip containing channels and reservoirs and (iii) an aqueous phase glass microarray for fluorescence microarray hybridization detection. The EWOD device offers the possibility of fully integrating on-chip sample preparation using nanolitre sample and reagent volumes. A key challenge is sample transfer from the oil phase EWOD device to the aqueous phase microarray for hybridization detection. The EWOD device, waveguide performance and functionality are maintained during the integration process. An on-chip biochemical protocol for arrayed primer extension (APEX) was implemented for single nucleotide polymorphism (SNiP) analysis. The prepared sample is aspirated from the EWOD oil phase to the aqueous phase microarray for hybridization. A bench-top instrumentation system was also developed around the integrated platform to drive the EWOD electrodes, implement APEX sample heating and image the microarray after hybridization.

Spacelab Charcoal Analyses

NASA Technical Reports Server (NTRS)

Slivon, L. E.; Hernon-Kenny, L. A.; Katona, V. R.; Dejarme, L. E.

1995-01-01

This report describes analytical methods and results obtained from chemical analysis of 31 charcoal samples in five sets. Each set was obtained from a single scrubber used to filter ambient air on board a Spacelab mission. Analysis of the charcoal samples was conducted by thermal desorption followed by gas chromatography/mass spectrometry (GC/MS). All samples were analyzed using identical methods. The method used for these analyses was able to detect compounds independent of their polarity or volatility. In addition to the charcoal samples, analyses of three Environmental Control and Life Support System (ECLSS) water samples were conducted specifically for trimethylamine.

Single-step solubilization of milk samples with N,N-dimethylformamide for inductively coupled plasma-mass spectrometry analysis and classification based on their elemental composition.

PubMed

Azcarate, Silvana M; Savio, Marianela; Smichowski, Patricia; Martinez, Luis D; Camiña, José M; Gil, Raúl A

2015-10-01

A single-step procedure for trace elements analysis of milk samples is presented. Solubilization with small amounts of dymethylformamide (DMF) was assayed prior to inductively coupled plasma mass spectrometry (ICPMS) detection with a high efficiency sample introduction system. All main instrumental conditions were optimized in order to readily introduce the samples without matrix elimination. In order to assess and mitigate matrix effects in the determination of As, Cd, Co, Cu, Eu, Ga, Gd, Ge, Mn, Mo, Nb, Nd, Ni, Pb, Pr, Rb, Sm, S, Sr, Ta, Tb, V, Zn, and Zr, matrix matching calibration with (103)Rh as internal standard (IS) was performed. The obtained limits of detection were between 0.68 (Tb) and 30 (Zn) μg L(-1). For accuracy verification, certified Skim milk powder reference material (BCR 063R) was employed. The developed method was applied to trace elements analysis of commercially available milks. Principal components analysis was used to correlate the content of trace metals with the kind of milk, obtaining a classification according to adults, baby or baby fortified milks. The outcomes highlight a simple and fast approach that could be trustworthy for routine analysis, quality control and traceability of milks. Copyright © 2015 Elsevier B.V. All rights reserved.

Forensic validation of the SNPforID 52-plex assay.

PubMed

Musgrave-Brown, Esther; Ballard, David; Balogh, Kinga; Bender, Klaus; Berger, Burkhard; Bogus, Magdalena; Børsting, Claus; Brion, María; Fondevila, Manuel; Harrison, Cheryl; Oguzturun, Ceylan; Parson, Walther; Phillips, Chris; Proff, Carsten; Ramos-Luis, Eva; Sanchez, Juan J; Sánchez Diz, Paula; Sobrino Rey, Bea; Stradmann-Bellinghausen, Beate; Thacker, Catherine; Carracedo, Angel; Morling, Niels; Scheithauer, Richard; Schneider, Peter M; Syndercombe Court, Denise

2007-06-01

The advantages of single nucleotide polymorphism (SNP) typing in forensic genetics are well known and include a wider choice of high-throughput typing platforms, lower mutation rates, and improved analysis of degraded samples. However, if SNPs are to become a realistic supplement to current short tandem repeat (STR) typing methods, they must be shown to successfully and reliably analyse the challenging samples commonly encountered in casework situations. The European SNPforID consortium, supported by the EU GROWTH programme, has developed a multiplex of 52 SNPs for forensic analysis, with the amplification of all 52 loci in a single reaction followed by two single base extension (SBE) reactions which are detected with capillary electrophoresis. In order to validate this assay, a variety of DNA extracts were chosen to represent problems such as low copy number and degradation that are commonly seen in forensic casework. A total of 40 extracts were used in the study, each of which was sent to two of the five participating laboratories for typing in duplicate or triplicate. Laboratories were instructed to carry out their analyses as if they were dealing with normal casework samples. Results were reported back to the coordinating laboratory and compared with those obtained from traditional STR typing of the same extracts using Powerplex 16 (Promega). These results indicate that, although the ability to successfully type good quality, low copy number extracts is lower, the 52-plex SNP assay performed better than STR typing on degraded samples, and also on samples that were both degraded and of limited quantity, suggesting that SNP analysis can provide advantages over STR analysis in forensically relevant circumstances. However, there were also additional problems arising from contamination and primer quality issues and these are discussed.

Use of Sequence-independent, single-primer amplification (SISPA) with NGS platform for detection of RNA viruses in clinical samples

USDA-ARS?s Scientific Manuscript database

Current technologies for next generation sequencing (NGS) have revolutionized metagenomics analysis of clinical samples. One advantage of the NGS platform is the possibility to sequence the genetic material in samples without any prior knowledge of the sequence contained within. Sequence-Independent...

Electrical and magnetic properties of nano-sized magnesium ferrite

NASA Astrophysics Data System (ADS)

T, Smitha; X, Sheena; J, Binu P.; Mohammed, E. M.

2015-02-01

Nano-sized magnesium ferrite was synthesized using sol-gel techniques. Structural characterization was done using X-ray diffractometer and Fourier Transform Infrared Spectrometer. Vibration Sample Magnetometer was used to record the magnetic measurements. XRD analysis reveals the prepared sample is single phasic without any impurity. Particle size calculation shows the average crystallite size of the sample is 19nm. FTIR analysis confirmed spinel structure of the prepared samples. Magnetic measurement study shows that the sample is ferromagnetic with high degree of isotropy. Hysterisis loop was traced at temperatures 100K and 300K. DC electrical resistivity measurements show semiconducting nature of the sample.

Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses

USDA-ARS?s Scientific Manuscript database

Current technologies with next generation sequencing have revolutionized metagenomics analysis of clinical samples. To achieve the non-selective amplification and recovery of low abundance genetic sequences, a simplified Sequence-Independent, Single-Primer Amplification (SISPA) technique in combinat...

Non-Destructive Study of Bulk Crystallinity and Elemental Composition of Natural Gold Single Crystal Samples by Energy-Resolved Neutron Imaging

PubMed Central

Tremsin, Anton S.; Rakovan, John; Shinohara, Takenao; Kockelmann, Winfried; Losko, Adrian S.; Vogel, Sven C.

2017-01-01

Energy-resolved neutron imaging enables non-destructive analyses of bulk structure and elemental composition, which can be resolved with high spatial resolution at bright pulsed spallation neutron sources due to recent developments and improvements of neutron counting detectors. This technique, suitable for many applications, is demonstrated here with a specific study of ~5–10 mm thick natural gold samples. Through the analysis of neutron absorption resonances the spatial distribution of palladium (with average elemental concentration of ~0.4 atom% and ~5 atom%) is mapped within the gold samples. At the same time, the analysis of coherent neutron scattering in the thermal and cold energy regimes reveals which samples have a single-crystalline bulk structure through the entire sample volume. A spatially resolved analysis is possible because neutron transmission spectra are measured simultaneously on each detector pixel in the epithermal, thermal and cold energy ranges. With a pixel size of 55 μm and a detector-area of 512 by 512 pixels, a total of 262,144 neutron transmission spectra are measured concurrently. The results of our experiments indicate that high resolution energy-resolved neutron imaging is a very attractive analytical technique in cases where other conventional non-destructive methods are ineffective due to sample opacity. PMID:28102285

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

PubMed

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

2012-01-03

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

Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture

PubMed Central

Bian, Shengtai; Cheng, Yinuo; Shi, Guanya; Liu, Peng; Ye, Xiongying

2017-01-01

Single cell analysis has received increasing attention recently in both academia and clinics, and there is an urgent need for effective upstream cell sample preparation. Two extremely challenging tasks in cell sample preparation—high-efficiency cell enrichment and precise single cell capture—have now entered into an era full of exciting technological advances, which are mostly enabled by microfluidics. In this review, we summarize the category of technologies that provide new solutions and creative insights into the two tasks of cell manipulation, with a focus on the latest development in the recent five years by highlighting the representative works. By doing so, we aim both to outline the framework and to showcase example applications of each task. In most cases for cell enrichment, we take circulating tumor cells (CTCs) as the target cells because of their research and clinical importance in cancer. For single cell capture, we review related technologies for many kinds of target cells because the technologies are supposed to be more universal to all cells rather than CTCs. Most of the mentioned technologies can be used for both cell enrichment and precise single cell capture. Each technology has its own advantages and specific challenges, which provide opportunities for researchers in their own area. Overall, these technologies have shown great promise and now evolve into real clinical applications. PMID:28217240

Applications of Single-Cell Sequencing for Multiomics.

PubMed

Xu, Yungang; Zhou, Xiaobo

2018-01-01

Single-cell sequencing interrogates the sequence or chromatin information from individual cells with advanced next-generation sequencing technologies. It provides a higher resolution of cellular differences and a better understanding of the underlying genetic and epigenetic mechanisms of an individual cell in the context of its survival and adaptation to microenvironment. However, it is more challenging to perform single-cell sequencing and downstream data analysis, owing to the minimal amount of starting materials, sample loss, and contamination. In addition, due to the picogram level of the amount of nucleic acids used, heavy amplification is often needed during sample preparation of single-cell sequencing, resulting in the uneven coverage, noise, and inaccurate quantification of sequencing data. All these unique properties raise challenges in and thus high demands for computational methods that specifically fit single-cell sequencing data. We here comprehensively survey the current strategies and challenges for multiple single-cell sequencing, including single-cell transcriptome, genome, and epigenome, beginning with a brief introduction to multiple sequencing techniques for single cells.

Single-Use Sensor Strips for Reliable Field Analysis of Gunshot Residue

DTIC Science & Technology

2013-10-13

3    List of Figures Fig. 1. Cyclic square-wave voltammogram at the bare GCE for a mixture of trace metals and explosives constituents of GSR: 3...variables obtained after CVA analysis of the GSR samples according to (B) exposure level or (C) 3-class response mode. Samples in (B) correspond to...to (A) exposure level or (B) 3-class response mode. Samples correspond to the same controls outlined in Fig. 6. Fig. 8. Score plot of the

Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy

PubMed Central

Actis, Paolo; Maalouf, Michelle; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R. Adam; Pourmand, Nader

2014-01-01

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis. PMID:24279711

DRUG-NEM: Optimizing drug combinations using single-cell perturbation response to account for intratumoral heterogeneity

PubMed Central

Anchang, Benedict; Davis, Kara L.; Fienberg, Harris G.; Bendall, Sean C.; Karacosta, Loukia G.; Tibshirani, Robert; Nolan, Garry P.; Plevritis, Sylvia K.

2018-01-01

An individual malignant tumor is composed of a heterogeneous collection of single cells with distinct molecular and phenotypic features, a phenomenon termed intratumoral heterogeneity. Intratumoral heterogeneity poses challenges for cancer treatment, motivating the need for combination therapies. Single-cell technologies are now available to guide effective drug combinations by accounting for intratumoral heterogeneity through the analysis of the signaling perturbations of an individual tumor sample screened by a drug panel. In particular, Mass Cytometry Time-of-Flight (CyTOF) is a high-throughput single-cell technology that enables the simultaneous measurements of multiple (>40) intracellular and surface markers at the level of single cells for hundreds of thousands of cells in a sample. We developed a computational framework, entitled Drug Nested Effects Models (DRUG-NEM), to analyze CyTOF single-drug perturbation data for the purpose of individualizing drug combinations. DRUG-NEM optimizes drug combinations by choosing the minimum number of drugs that produce the maximal desired intracellular effects based on nested effects modeling. We demonstrate the performance of DRUG-NEM using single-cell drug perturbation data from tumor cell lines and primary leukemia samples. PMID:29654148

Detection of clonal evolution in hematopoietic malignancies by combining comparative genomic hybridization and single nucleotide polymorphism arrays.

PubMed

Hartmann, Luise; Stephenson, Christine F; Verkamp, Stephanie R; Johnson, Krystal R; Burnworth, Bettina; Hammock, Kelle; Brodersen, Lisa Eidenschink; de Baca, Monica E; Wells, Denise A; Loken, Michael R; Zehentner, Barbara K

2014-12-01

Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies. Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample's clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies. Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated. © 2014 American Association for Clinical Chemistry.

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

EPA Science Inventory

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

Population transcriptomics with single-cell resolution: a new field made possible by microfluidics: a technology for high throughput transcript counting and data-driven definition of cell types.

PubMed

Plessy, Charles; Desbois, Linda; Fujii, Teruo; Carninci, Piero

2013-02-01

Tissues contain complex populations of cells. Like countries, which are comprised of mixed populations of people, tissues are not homogeneous. Gene expression studies that analyze entire populations of cells from tissues as a mixture are blind to this diversity. Thus, critical information is lost when studying samples rich in specialized but diverse cells such as tumors, iPS colonies, or brain tissue. High throughput methods are needed to address, model and understand the constitutive and stochastic differences between individual cells. Here, we describe microfluidics technologies that utilize a combination of molecular biology and miniaturized labs on chips to study gene expression at the single cell level. We discuss how the characterization of the transcriptome of each cell in a sample will open a new field in gene expression analysis, population transcriptomics, that will change the academic and biomedical analysis of complex samples by defining them as quantified populations of single cells. Copyright © 2013 WILEY Periodicals, Inc.

Non-invasive optoacoustic probing of the density and stiffness of single biological cells

NASA Astrophysics Data System (ADS)

Dehoux, T.; Audoin, B.

2012-12-01

Recently, the coherent generation of GHz acoustic waves using ultrashort laser pulses has demonstrated the ability to probe the sound velocity in vegetal cells and in cell-mimicking soft micro-objects with micrometer resolution, opening tremendous potentialities for single-cell biology. However, manipulating biological media in physiological conditions is often a technical challenge when using a laser-based setup. In this article, we present a new opto-acoustic bio-transducer composed of a thin metal film sputtered on a transparent heat sink that allows reducing importantly the laser-induced cellular stresses, and offers a wide variety of optical configurations. In particular, by exploiting the acoustic reflection coefficient at the sample-transducer interface and the photoacoustic interaction inside the transparent sample, the density and compressibility of the sample can be probed simultaneously. Using an ad hoc signal analysis based on Hilbert and wavelet transforms, these quantities are measured accurately for a reference fluid. Similar analysis performed in a single vegetal cell also suggests high sensitivity to the state of the transducer-cell interface, and notably to the presence of the plasma membrane that encloses the cell vacuole.

Trajectory Design for a Single-String Impactor Concept

NASA Technical Reports Server (NTRS)

Dono Perez, Andres; Burton, Roland; Stupl, Jan; Mauro, David

2017-01-01

This paper introduces a trajectory design for a secondary spacecraft concept to augment science return in interplanetary missions. The concept consist of a single-string probe with a kinetic impactor on board that generates an artificial plume to perform in-situ sampling. The trajectory design was applied to a particular case study that samples ejecta particles from the Jovian moon Europa. Results were validated using statistical analysis. Details regarding the navigation, targeting and disposal challenges related to this concept are presented herein.

Analysis report for 241-BY-104 Auger samples

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

Beck, M.A.

1994-11-10

This report describes the analysis of the surface crust samples taken from single-shell tank (SST) BY-104, suspected of containing ferrocyanide wastes. This sampling and analysis will assist in ascertaining whether there is any hazard due to combustion (burning) or explosion of these solid wastes. These characteristics are important to future efforts to characterize the salt and sludge in this type of waste tank. This report will outline the methodology and detail the results of analyses performed during the characterization of this material. All analyses were performed by Westinghouse Hanford Company at the 222-S laboratory unless stated otherwise.

Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties

NASA Astrophysics Data System (ADS)

Schmid, Sonja; Hugel, Thorsten

2018-03-01

Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings—such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.

Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors.

PubMed

Lukeš, Tomáš; Pospíšil, Jakub; Fliegel, Karel; Lasser, Theo; Hagen, Guy M

2018-03-01

Super-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared with organic dyes, which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms. Four complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented, including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM datasets using a different method: super-resolution optical fluctuation imaging (SOFI). The 2 modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes. This dataset has potential for extensive reuse. Complete raw data from SMLM experiments have typically not been published. The YFP data exhibit low signal-to-noise ratios, making data analysis a challenge. These datasets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

[Influence of sample surface roughness on mathematical model of NIR quantitative analysis of wood density].

PubMed

Huang, An-Min; Fei, Ben-Hua; Jiang, Ze-Hui; Hse, Chung-Yun

2007-09-01

Near infrared spectroscopy is widely used as a quantitative method, and the main multivariate techniques consist of regression methods used to build prediction models, however, the accuracy of analysis results will be affected by many factors. In the present paper, the influence of different sample roughness on the mathematical model of NIR quantitative analysis of wood density was studied. The result of experiments showed that if the roughness of predicted samples was consistent with that of calibrated samples, the result was good, otherwise the error would be much higher. The roughness-mixed model was more flexible and adaptable to different sample roughness. The prediction ability of the roughness-mixed model was much better than that of the single-roughness model.

Integrated Multi-process Microfluidic Systems for Automating Analysis

PubMed Central

Yang, Weichun; Woolley, Adam T.

2010-01-01

Microfluidic technologies have been applied extensively in rapid sample analysis. Some current challenges for standard microfluidic systems are relatively high detection limits, and reduced resolving power and peak capacity compared to conventional approaches. The integration of multiple functions and components onto a single platform can overcome these separation and detection limitations of microfluidics. Multiplexed systems can greatly increase peak capacity in multidimensional separations and can increase sample throughput by analyzing many samples simultaneously. On-chip sample preparation, including labeling, preconcentration, cleanup and amplification, can all serve to speed up and automate processes in integrated microfluidic systems. This paper summarizes advances in integrated multi-process microfluidic systems for automated analysis, their benefits and areas for needed improvement. PMID:20514343

A LDR-PCR approach for multiplex polymorphisms genotyping of severely degraded DNA with fragment sizes <100 bp.

PubMed

Zhang, Zhen; Wang, Bao-Jie; Guan, Hong-Yu; Pang, Hao; Xuan, Jin-Feng

2009-11-01

Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini-short tandem repeat-polymerase chain reaction (PCR) and mini-sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small-scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.

Free-breathing Sparse Sampling Cine MR Imaging with Iterative Reconstruction for the Assessment of Left Ventricular Function and Mass at 3.0 T.

PubMed

Sudarski, Sonja; Henzler, Thomas; Haubenreisser, Holger; Dösch, Christina; Zenge, Michael O; Schmidt, Michaela; Nadar, Mariappan S; Borggrefe, Martin; Schoenberg, Stefan O; Papavassiliu, Theano

2017-01-01

Purpose To prospectively evaluate the accuracy of left ventricle (LV) analysis with a two-dimensional real-time cine true fast imaging with steady-state precession (trueFISP) magnetic resonance (MR) imaging sequence featuring sparse data sampling with iterative reconstruction (SSIR) performed with and without breath-hold (BH) commands at 3.0 T. Materials and Methods Ten control subjects (mean age, 35 years; range, 25-56 years) and 60 patients scheduled to undergo a routine cardiac examination that included LV analysis (mean age, 58 years; range, 20-86 years) underwent a fully sampled segmented multiple BH cine sequence (standard of reference) and a prototype undersampled SSIR sequence performed during a single BH and during free breathing (non-BH imaging). Quantitative analysis of LV function and mass was performed. Linear regression, Bland-Altman analysis, and paired t testing were performed. Results Similar to the results in control subjects, analysis of the 60 patients showed excellent correlation with the standard of reference for single-BH SSIR (r = 0.93-0.99) and non-BH SSIR (r = 0.92-0.98) for LV ejection fraction (EF), volume, and mass (P < .0001 for all). Irrespective of breath holding, LV end-diastolic mass was overestimated with SSIR (standard of reference: 163.9 g ± 58.9, single-BH SSIR: 178.5 g ± 62.0 [P < .0001], non-BH SSIR: 175.3 g ± 63.7 [P < .0001]); the other parameters were not significantly different (EF: 49.3% ± 11.9 with standard of reference, 48.8% ± 11.8 with single-BH SSIR, 48.8% ± 11 with non-BH SSIR; P = .03 and P = .12, respectively). Bland-Altman analysis showed similar measurement errors for single-BH SSIR and non-BH SSIR when compared with standard of reference measurements for EF, volume, and mass. Conclusion Assessment of LV function with SSIR at 3.0 T is noninferior to the standard of reference irrespective of BH commands. LV mass, however, is overestimated with SSIR. © RSNA, 2016 Online supplemental material is available for this article.

Is It Attachment Style or Socio-Demography: Singlehood in a Representative Sample.

PubMed

Petrowski, Katja; Schurig, Susan; Schmutzer, Gabriele; Brähler, Elmar; Stöbel-Richter, Yve

2015-01-01

Since the percentage of single adults is steadily increasing, the reasons for this development have become a matter of growing interest. Hereby, an individual's attachment style may have a connection to the partnership status. In the following analysis, attachment style, gender, age, education, and income were compared in regard to the partnership status. Furthermore, an analysis of variance was computed to compare the attachment style within different groups. In 2012, a sample of 1,676 representative participants was used. The participants were aged 18 to 60 (M = 41.0, SD = 12.3); 54% of the sample were female, and 40% were single. Attachment-related attitudes were assessed with the German version of the adult attachment scale (AAS). Single adult males did not show a more anxious attachment style than single adult females or females in relationships. Younger, i.e., 18 to 30 years old, paired individuals showed greater attachment anxiety than single individuals, whereby single individuals between the ages of 31 to 45 showed greater attachment anxiety than individuals in relationships. In addition, single individuals more frequently had obtained their high school diploma in contrast to individuals in relationships. Concerning attachment style, the individuals who had not completed their high school diploma showed less faith in others independent of singlehood or being in a relationship. Concerning age, older single individuals, i.e., 46 to 60 years, felt less comfortable in respect to closeness and showed less faith in others compared to paired individuals. Logistic regression showed that individuals were not single if they did not mind depending on others, showed high attachment anxiety, were older, and had lower education. An income below € 2000/month was linked to a nearly 13-fold increase of likelihood of being single. In sum, the attachment style had a differential age-dependent association to singlehood versus being in a relationship. Education played also a role, exclusively concerning faith in others.

Development of a simultaneous multiple solid-phase microextraction-single shot-gas chromatography/mass spectrometry method and application to aroma profile analysis of commercial coffee.

PubMed

Lee, Changgook; Lee, Younghoon; Lee, Jae-Gon; Buglass, Alan J

2013-06-21

A simultaneous multiple solid-phase microextraction-single shot-gas chromatography mass spectrometry (smSPME-ss-GC/MS) method has been developed for headspace analysis. Up to four fibers (50/30 μm DVB/CAR/PDMS) were used simultaneously for the extraction of aroma components from the headspace of a single sample chamber in order to increase sensitivity of aroma extraction. To avoid peak broadening and to maximize resolution, a simple cryofocusing technique was adopted during sequential thermal desorption of multiple SPME fibers prior to a 'single shot' chromatographic run. The method was developed and validated on a model flavor mixture, containing 81 known pure components. With the conditions of 10 min of incubation and 30 min of extraction at 50 °C, single, dual, triple and quadruple SPME extractions were compared. The increase in total peak area with increase in the number of fibers showed good linearity (R(2)=0.9917) and the mean precision was 12.0% (RSD) for the total peak sum, with quadruple simultaneous SPME extraction. Using a real sample such as commercial coffee granules, aroma profile analysis was conducted using single, dual, triple and quadruple SPME fibers. The increase in total peak intensity again showed good linearity with increase in the number of SPME fibers used (R(2)=0.9992) and the precision of quadruple SPME extraction was 9.9% (RSD) for the total peak sum. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

PubMed

Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

1998-10-01

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

Association of HS6ST3 gene polymorphisms with obesity and triglycerides: gene x gender interaction.

PubMed

Wang, Ke-Sheng; Wang, Liang; Liu, Xuefeng; Zeng, Min

2013-12-01

The heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) gene is involved in heparan sulphate and heparin metabolism, and has been reported to be associated with diabetic retinopathy in type 2 diabetes.We hypothesized that HS6ST3 gene polymorphisms might play an important role in obesity and related phenotypes (such as triglycerides). We examined genetic associations of 117 single-nucleotide polymorphisms (SNPs) within the HS6ST3 gene with obesity and triglycerides using two Caucasian samples: the Marshfield sample (1442 obesity cases and 2122 controls), and the Health aging and body composition (Health ABC) sample (305 cases and 1336 controls). Logistic regression analysis of obesity as a binary trait and linear regression analysis of triglycerides as a continuous trait, adjusted for age and sex, were performed using PLINK. Single marker analysis showed that six SNPs in the Marshfield sample and one SNP in the Health ABC sample were associated with obesity (P < 0.05). SNP rs535812 revealed a stronger association with obesity in meta-analysis of these two samples (P = 0.0105). The T-A haplotype from rs878950 and rs9525149 revealed significant association with obesity in the Marshfield sample (P = 0.012). Moreover, nine SNPs showed associations with triglycerides in the Marshfield sample (P < 0.05) and the best signal was rs1927796 (P = 0.00858). In addition, rs7331762 showed a strong gene x gender interaction (P = 0.00956) for obesity while rs1927796 showed a strong gene x gender interaction (P = 0.000625) for triglycerides in the Marshfield sample. These findings contribute new insights into the pathogenesis of obesity and triglycerides and demonstrate the importance of gender differences in the aetiology.

High-throughput microfluidic single-cell digital polymerase chain reaction.

PubMed

White, A K; Heyries, K A; Doolin, C; Vaninsberghe, M; Hansen, C L

2013-08-06

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

Being Single as a Social Barrier to Access Reproductive Healthcare Services by Iranian Girls

PubMed Central

Kohan, Shahnaz; Mohammadi, Fatemeh; Mostafavi, Firoozeh; Gholami, Ali

2017-01-01

Background: Iranian single women are deprived of reproductive healthcare services, though the provision of such services to the public has increased. This study aimed to explore the experiences of Iranian single women on their access to reproductive health services. Methods: A qualitative design using a conventional content analysis method was used. Semi-structured interviews were held with 17 single women and nine health providers chosen using the purposive sampling method. Results: Data analysis resulted in the development of three categories: ‘family’s attitudes and performance about single women’s reproductive healthcare,’ ‘socio-cultural factors influencing reproductive healthcare,’ and ‘cultural factors influencing being a single woman.’ Conclusion: Cultural and contextual factors affect being a single woman in every society. Therefore, healthcare providers need to identify such factors during the designing of strategies for improving the facilitation of access to reproductive healthcare services. PMID:28812794

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

Madden, Jeremy T.; Toth, Scott J.; Dettmar, Christopher M.

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ~10 3–10 4-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering andmore » analysis of phenylalanine hydroxylase fromChromobacterium violaceumcPAH,Trichinella spiralisdeubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.« less

PROP3D: A Program for 3D Euler Unsteady Aerodynamic and Aeroelastic (Flutter and Forced Response) Analysis of Propellers. Version 1.0

NASA Technical Reports Server (NTRS)

Srivastava, R.; Reddy, T. S. R.

1996-01-01

This guide describes the input data required, for steady or unsteady aerodynamic and aeroelastic analysis of propellers and the output files generated, in using PROP3D. The aerodynamic forces are obtained by solving three dimensional unsteady, compressible Euler equations. A normal mode structural analysis is used to obtain the aeroelastic equations, which are solved using either time domain or frequency domain solution method. Sample input and output files are included in this guide for steady aerodynamic analysis of single and counter-rotation propellers, and aeroelastic analysis of single-rotation propeller.

Detection of Babesia canis vogeli and Hepatozoon canis in canine blood by a single-tube real-time fluorescence resonance energy transfer polymerase chain reaction assay and melting curve analysis.

PubMed

Kongklieng, Amornmas; Intapan, Pewpan M; Boonmars, Thidarut; Thanchomnang, Tongjit; Janwan, Penchom; Sanpool, Oranuch; Lulitanond, Viraphong; Taweethavonsawat, Piyanan; Chungpivat, Sudchit; Maleewong, Wanchai

2015-03-01

A real-time fluorescence resonance energy transfer polymerase chain reaction (qFRET PCR) coupled with melting curve analysis was developed for detection of Babesia canis vogeli and Hepatozoon canis infections in canine blood samples in a single tube assay. The target of the assay was a region within the 18S ribosomal RNA gene amplified in either species by a single pair of primers. Following amplification from the DNA of infected dog blood, a fluorescence melting curve analysis was done. The 2 species, B. canis vogeli and H. canis, could be detected and differentiated in infected dog blood samples (n = 37) with high sensitivity (100%). The detection limit for B. canis vogeli was 15 copies of a positive control plasmid, and for H. canis, it was 150 copies of a positive control plasmid. The assay could simultaneously distinguish the DNA of both parasites from the DNA of controls. Blood samples from 5 noninfected dogs were negative, indicating high specificity. Several samples can be run at the same time. The assay can reduce misdiagnosis and the time associated with microscopic examination, and is not prone to the carryover contamination associated with the agarose gel electrophoresis step of conventional PCR. In addition, this qFRET PCR method would be useful to accurately determine the range of endemic areas or to discover those areas where the 2 parasites co-circulate. © 2015 The Author(s).

Combining band recovery data and Pollock's robust design to model temporary and permanent emigration

USGS Publications Warehouse

Lindberg, M.S.; Kendall, W.L.; Hines, J.E.; Anderson, M.G.

2001-01-01

Capture-recapture models are widely used to estimate demographic parameters of marked populations. Recently, this statistical theory has been extended to modeling dispersal of open populations. Multistate models can be used to estimate movement probabilities among subdivided populations if multiple sites are sampled. Frequently, however, sampling is limited to a single site. Models described by Burnham (1993, in Marked Individuals in the Study of Bird Populations, 199-213), which combined open population capture-recapture and band-recovery models, can be used to estimate permanent emigration when sampling is limited to a single population. Similarly, Kendall, Nichols, and Hines (1997, Ecology 51, 563-578) developed models to estimate temporary emigration under Pollock's (1982, Journal of Wildlife Management 46, 757-760) robust design. We describe a likelihood-based approach to simultaneously estimate temporary and permanent emigration when sampling is limited to a single population. We use a sampling design that combines the robust design and recoveries of individuals obtained immediately following each sampling period. We present a general form for our model where temporary emigration is a first-order Markov process, and we discuss more restrictive models. We illustrate these models with analysis of data on marked Canvasback ducks. Our analysis indicates that probability of permanent emigration for adult female Canvasbacks was 0.193 (SE = 0.082) and that birds that were present at the study area in year i - 1 had a higher probability of presence in year i than birds that were not present in year i - 1.

Computationally Aided Absolute Stereochemical Determination of Enantioenriched Amines.

PubMed

Zhang, Jun; Gholami, Hadi; Ding, Xinliang; Chun, Minji; Vasileiou, Chrysoula; Nehira, Tatsuo; Borhan, Babak

2017-03-17

A simple and efficient protocol for sensing the absolute stereochemistry and enantiomeric excess of chiral monoamines is reported. Preparation of the sample requires a single-step reaction of the 1,1'-(bromomethylene)dinaphthalene (BDN) with the chiral amine. Analysis of the exciton coupled circular dichroism generated from the BDN-derivatized chiral amine sample, along with comparison to conformational analysis performed computationally, yields the absolute stereochemistry of the parent chiral monoamine.

Target capture enrichment of nuclear SNP markers for massively parallel sequencing of degraded and mixed samples.

PubMed

Bose, Nikhil; Carlberg, Katie; Sensabaugh, George; Erlich, Henry; Calloway, Cassandra

2018-05-01

DNA from biological forensic samples can be highly fragmented and present in limited quantity. When DNA is highly fragmented, conventional PCR based Short Tandem Repeat (STR) analysis may fail as primer binding sites may not be present on a single template molecule. Single Nucleotide Polymorphisms (SNPs) can serve as an alternative type of genetic marker for analysis of degraded samples because the targeted variation is a single base. However, conventional PCR based SNP analysis methods still require intact primer binding sites for target amplification. Recently, probe capture methods for targeted enrichment have shown success in recovering degraded DNA as well as DNA from ancient bone samples using next-generation sequencing (NGS) technologies. The goal of this study was to design and test a probe capture assay targeting forensically relevant nuclear SNP markers for clonal and massively parallel sequencing (MPS) of degraded and limited DNA samples as well as mixtures. A set of 411 polymorphic markers totaling 451 nuclear SNPs (375 SNPs and 36 microhaplotype markers) was selected for the custom probe capture panel. The SNP markers were selected for a broad range of forensic applications including human individual identification, kinship, and lineage analysis as well as for mixture analysis. Performance of the custom SNP probe capture NGS assay was characterized by analyzing read depth and heterozygote allele balance across 15 samples at 25 ng input DNA. Performance thresholds were established based on read depth ≥500X and heterozygote allele balance within ±10% deviation from 50:50, which was observed for 426 out of 451 SNPs. These 426 SNPs were analyzed in size selected samples (at ≤75 bp, ≤100 bp, ≤150 bp, ≤200 bp, and ≤250 bp) as well as mock degraded samples fragmented to an average of 150 bp. Samples selected for ≤75 bp exhibited 99-100% reportable SNPs across varied DNA amounts and as low as 0.5 ng. Mock degraded samples at 1 ng and 10 ng exhibited >90% reportable SNPs. Finally, two-person male-male mixtures were tested at 10 ng in contributor varying ratios. Overall, 85-100% of alleles unique to the minor contributor were observed at all mixture ratios. Results from these studies using the SNP probe capture NGS system demonstrates proof of concept for application to forensically relevant degraded and mixed DNA samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of Variance with Summary Statistics in Microsoft® Excel®

ERIC Educational Resources Information Center

Larson, David A.; Hsu, Ko-Cheng

2010-01-01

Students regularly are asked to solve Single Factor Analysis of Variance problems given only the sample summary statistics (number of observations per category, category means, and corresponding category standard deviations). Most undergraduate students today use Excel for data analysis of this type. However, Excel, like all other statistical…

Digital DNA detection based on a compact optofluidic laser with ultra-low sample consumption.

PubMed

Lee, Wonsuk; Chen, Qiushu; Fan, Xudong; Yoon, Dong Ki

2016-11-29

DNA lasers self-amplify optical signals from a DNA analyte as well as thermodynamic differences between sequences, allowing quasi-digital DNA detection. However, these systems have drawbacks, such as relatively large sample consumption and complicated dye labelling. Moreover, although the lasing signal can detect the target DNA, it is superimposed on an unintended fluorescence background, which persists for non-target DNA samples as well. From an optical point of view, it is thus not truly digital detection and requires spectral analysis to identify the target. In this work, we propose and demonstrate an optofluidic laser that has a single layer of DNA molecules as the gain material. A target DNA produces intensive laser emission comparable to existing DNA lasers, while any unnecessary fluorescence background is successfully suppressed. As a result, the target DNA can be detected with a single laser pulse, in a truly digital manner. Since the DNA molecules cover only a single layer on the surface of the laser microcavity, the DNA sample consumption is a few orders of magnitude lower than that of existing DNA lasers. Furthermore, the DNA molecules are stained by simply immersing the microcavity in the intercalating dye solution, and thus the proposed DNA laser is free of any complex dye-labelling process prior to analysis.

Improvements in the gaseous hydrogen-water equilibration technique for hydrogen isotope ratio analysis

USGS Publications Warehouse

Coplen, T.B.; Wildman, J.D.; Chen, J.

1991-01-01

Improved precision in the H2-H2O equilibration method for ??D analysis has been achieved in an automated system. Reduction in 1-?? standard deviation of a single mass-spectrometer analysis to 1.3??? is achieved by (1) bonding catalyst to glass rods and assigning use to specific equilibration chambers to monitor performance of catalyst, (2) improving the apparatus design, and (3) reducing the H3+ contribution of the mass-spectrometer ion source. For replicate analysis of a water sample, the standard deviation improved to 0.8???. H2S-bearing samples and samples as small as 0.1 mL can be analyzed routinely with this method.

Evaluation of targeted exome sequencing for 28 protein-based blood group systems, including the homologous gene systems, for blood group genotyping.

PubMed

Schoeman, Elizna M; Lopez, Genghis H; McGowan, Eunike C; Millard, Glenda M; O'Brien, Helen; Roulis, Eileen V; Liew, Yew-Wah; Martin, Jacqueline R; McGrath, Kelli A; Powley, Tanya; Flower, Robert L; Hyland, Catherine A

2017-04-01

Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS. Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting. © 2017 AABB.

Granatum: a graphical single-cell RNA-Seq analysis pipeline for genomics scientists.

PubMed

Zhu, Xun; Wolfgruber, Thomas K; Tasato, Austin; Arisdakessian, Cédric; Garmire, David G; Garmire, Lana X

2017-12-05

Single-cell RNA sequencing (scRNA-Seq) is an increasingly popular platform to study heterogeneity at the single-cell level. Computational methods to process scRNA-Seq data are not very accessible to bench scientists as they require a significant amount of bioinformatic skills. We have developed Granatum, a web-based scRNA-Seq analysis pipeline to make analysis more broadly accessible to researchers. Without a single line of programming code, users can click through the pipeline, setting parameters and visualizing results via the interactive graphical interface. Granatum conveniently walks users through various steps of scRNA-Seq analysis. It has a comprehensive list of modules, including plate merging and batch-effect removal, outlier-sample removal, gene-expression normalization, imputation, gene filtering, cell clustering, differential gene expression analysis, pathway/ontology enrichment analysis, protein network interaction visualization, and pseudo-time cell series construction. Granatum enables broad adoption of scRNA-Seq technology by empowering bench scientists with an easy-to-use graphical interface for scRNA-Seq data analysis. The package is freely available for research use at http://garmiregroup.org/granatum/app.

Hierarchical Linear Modeling Meta-Analysis of Single-Subject Design Research

ERIC Educational Resources Information Center

Gage, Nicholas A.; Lewis, Timothy J.

2014-01-01

The identification of evidence-based practices continues to provoke issues of disagreement across multiple fields. One area of contention is the role of single-subject design (SSD) research in providing scientific evidence. The debate about SSD's utility centers on three issues: sample size, effect size, and serial dependence. One potential…

A New Method Using Single-Particle Mass Spectrometry Data to Distinguish Mineral Dust and Biological Aerosols

NASA Astrophysics Data System (ADS)

Al-Mashat, H.; Kristensen, L.; Sultana, C. M.; Prather, K. A.

2016-12-01

The ability to distinguish types of particles present within a cloud is important for determining accurate inputs to climate models. The chemical composition of particles within cloud liquid droplets and ice crystals can have a significant impact on the timing, location, and amount of precipitation that falls. Precipitation efficiency is increased by the presence of ice crystals in clouds, and both mineral dust and biological aerosols have been shown to be effective ice nucleating particles (INPs) in the atmosphere. A current challenge in aerosol science is distinguishing mineral dust and biological material in the analysis of real-time, ambient, single-particle mass spectral data. Single-particle mass spectrometers are capable of measuring the size-resolved chemical composition of individual atmospheric particles. However, there is no consistent analytical method for distinguishing dust and biological aerosols. Sampling and characterization of control samples (i.e. of known identity) of mineral dust and bacteria were performed by the Aerosol Time-of-Flight Mass Spectrometer (ATOFMS) as part of the Fifth Ice Nucleation (FIN01) Workshop at the Aerosol Interaction and Dynamics in the Atmosphere (AIDA) facility in Karlsruhe, Germany. Using data collected by the ATOFMS of control samples, a new metric has been developed to classify single particles as dust or biological independent of spectral cluster analysis. This method, involving the use of a ratio of mass spectral peak areas for organic nitrogen and silicates, is easily reproducible and does not rely on extensive knowledge of particle chemistry or the ionization characteristics of mass spectrometers. This represents a step toward rapidly distinguishing particle types responsible for ice nucleation activity during real-time sampling in clouds. The ability to distinguish types of particles present within a cloud is important for determining accurate inputs to climate models. The chemical composition of particles within cloud liquid droplets and ice crystals can have a significant impact on the timing, location, and amount of precipitation that falls. Precipitation efficiency is increased by the presence of ice crystals in clouds, and both mineral dust and biological aerosols have been shown to be effective ice nucleating particles (INPs) in the atmosphere. A current challenge in aerosol science is distinguishing mineral dust and biological material in the analysis of real-time, ambient, single-particle mass spectral data. Single-particle mass spectrometers are capable of measuring the size-resolved chemical composition of individual atmospheric particles. However, there is no consistent analytical method for distinguishing dust and biological aerosols. Sampling and characterization of control samples (i.e. of known identity) of mineral dust and bacteria were performed by the Aerosol Time-of-Flight Mass Spectrometer (ATOFMS) as part of the Fifth Ice Nucleation (FIN01) Workshop at the Aerosol Interaction and Dynamics in the Atmosphere (AIDA) facility in Karlsruhe, Germany. Using data collected by the ATOFMS of control samples, a new metric has been developed to classify single particles as dust or biological independent of spectral cluster analysis. This method, involving the use of a ratio of mass spectral peak areas for organic nitrogen and silicates, is easily reproducible and does not rely on extensive knowledge of particle chemistry or the ionization characteristics of mass spectrometers. This represents a step toward rapidly distinguishing particle types responsible for ice nucleation activity during real-time sampling in clouds.

Surface Analysis of Nerve Agent Degradation Products by ...

EPA Pesticide Factsheets

Report This sampling and analytical procedure was developed and applied by a single laboratory to investigate nerve agent degradation products, which may persist at a contaminated site, via surface wiping followed by analytical characterization. The performance data presented demonstrate the fitness-for-purpose regarding surface analysis in that single laboratory. Surfaces (laminate, glass, galvanized steel, vinyl tile, painted drywall and treated wood) were wiped with cotton gauze wipes, sonicated, extracted with distilled water, and filtered. Samples were analyzed with direct injection electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS) without derivatization. Detection limit data were generated for all analytes of interest on a laminate surface. Accuracy and precision data were generated from each surface fortified with these analytes.

A multitask clustering approach for single-cell RNA-seq analysis in Recessive Dystrophic Epidermolysis Bullosa

PubMed Central

Petegrosso, Raphael; Tolar, Jakub

2018-01-01

Single-cell RNA sequencing (scRNA-seq) has been widely applied to discover new cell types by detecting sub-populations in a heterogeneous group of cells. Since scRNA-seq experiments have lower read coverage/tag counts and introduce more technical biases compared to bulk RNA-seq experiments, the limited number of sampled cells combined with the experimental biases and other dataset specific variations presents a challenge to cross-dataset analysis and discovery of relevant biological variations across multiple cell populations. In this paper, we introduce a method of variance-driven multitask clustering of single-cell RNA-seq data (scVDMC) that utilizes multiple single-cell populations from biological replicates or different samples. scVDMC clusters single cells in multiple scRNA-seq experiments of similar cell types and markers but varying expression patterns such that the scRNA-seq data are better integrated than typical pooled analyses which only increase the sample size. By controlling the variance among the cell clusters within each dataset and across all the datasets, scVDMC detects cell sub-populations in each individual experiment with shared cell-type markers but varying cluster centers among all the experiments. Applied to two real scRNA-seq datasets with several replicates and one large-scale droplet-based dataset on three patient samples, scVDMC more accurately detected cell populations and known cell markers than pooled clustering and other recently proposed scRNA-seq clustering methods. In the case study applied to in-house Recessive Dystrophic Epidermolysis Bullosa (RDEB) scRNA-seq data, scVDMC revealed several new cell types and unknown markers validated by flow cytometry. MATLAB/Octave code available at https://github.com/kuanglab/scVDMC. PMID:29630593

Zooplankton community analysis in the Changjiang River estuary by single-gene-targeted metagenomics

NASA Astrophysics Data System (ADS)

Cheng, Fangping; Wang, Minxiao; Li, Chaolun; Sun, Song

2014-07-01

DNA barcoding provides accurate identification of zooplankton species through all life stages. Single-gene-targeted metagenomic analysis based on DNA barcode databases can facilitate longterm monitoring of zooplankton communities. With the help of the available zooplankton databases, the zooplankton community of the Changjiang (Yangtze) River estuary was studied using a single-gene-targeted metagenomic method to estimate the species richness of this community. A total of 856 mitochondrial cytochrome oxidase subunit 1 (cox1) gene sequences were determined. The environmental barcodes were clustered into 70 molecular operational taxonomic units (MOTUs). Forty-two MOTUs matched barcoded marine organisms with more than 90% similarity and were assigned to either the species (similarity>96%) or genus level (similarity<96%). Sibling species could also be distinguished. Many species that were overlooked by morphological methods were identified by molecular methods, especially gelatinous zooplankton and merozooplankton that were likely sampled at different life history phases. Zooplankton community structures differed significantly among all of the samples. The MOTU spatial distributions were influenced by the ecological habits of the corresponding species. In conclusion, single-gene-targeted metagenomic analysis is a useful tool for zooplankton studies, with which specimens from all life history stages can be identified quickly and effectively with a comprehensive database.

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry.

PubMed

Zhu, Hongying; Zou, Guichang; Wang, Ning; Zhuang, Meihui; Xiong, Wei; Huang, Guangming

2017-03-07

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules.

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry

PubMed Central

Zhu, Hongying; Zou, Guichang; Wang, Ning; Zhuang, Meihui; Xiong, Wei; Huang, Guangming

2017-01-01

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules. PMID:28223513

Single-Case Experimental Designs: A Systematic Review of Published Research and Current Standards

PubMed Central

Smith, Justin D.

2013-01-01

This article systematically reviews the research design and methodological characteristics of single-case experimental design (SCED) research published in peer-reviewed journals between 2000 and 2010. SCEDs provide researchers with a flexible and viable alternative to group designs with large sample sizes. However, methodological challenges have precluded widespread implementation and acceptance of the SCED as a viable complementary methodology to the predominant group design. This article includes a description of the research design, measurement, and analysis domains distinctive to the SCED; a discussion of the results within the framework of contemporary standards and guidelines in the field; and a presentation of updated benchmarks for key characteristics (e.g., baseline sampling, method of analysis), and overall, it provides researchers and reviewers with a resource for conducting and evaluating SCED research. The results of the systematic review of 409 studies suggest that recently published SCED research is largely in accordance with contemporary criteria for experimental quality. Analytic method emerged as an area of discord. Comparison of the findings of this review with historical estimates of the use of statistical analysis indicates an upward trend, but visual analysis remains the most common analytic method and also garners the most support amongst those entities providing SCED standards. Although consensus exists along key dimensions of single-case research design and researchers appear to be practicing within these parameters, there remains a need for further evaluation of assessment and sampling techniques and data analytic methods. PMID:22845874

Obsidian sources characterized by neutron-activation analysis.

PubMed

Gordus, A A; Wright, G A; Griffin, J B

1968-07-26

Concentrations of elements such as manganese, scandium, lanthanum, rubidium, samarium, barium, and zirconium in obsidian samples from different flows show ranges of 1000 percent or more, whereas the variation in element content in obsidian samples from a single flow appears to be less than 40 percent. Neutron-activation analysis of these elements, as well as of sodium and iron, provides a means of identifying the geologic source of an archeological artifact of obsidian.

A METHOD FOR AUTOMATED ANALYSIS OF 10 ML WATER SAMPLES CONTAINING ACIDIC, BASIC, AND NEUTRAL SEMIVOLATILE COMPOUNDS LISTED IN USEPA METHOD 8270 BY SOLID PHASE EXTRACTION COUPLED IN-LINE TO LARGE VOLUME INJECTION GAS CHROMATOGRAPHY/MASS SPECTROMETRY

EPA Science Inventory

Data is presented showing the progress made towards the development of a new automated system combining solid phase extraction (SPE) with gas chromatography/mass spectrometry for the single run analysis of water samples containing a broad range of acid, base and neutral compounds...

Diversity changes of microbial communities into hospital surface environments.

PubMed

Yano, Rika; Shimoda, Tomoko; Watanabe, Reina; Kuroki, Yasutoshi; Okubo, Torahiko; Nakamura, Shinji; Matsuo, Junji; Yoshimura, Sadako; Yamaguchi, Hiroyuki

2017-07-01

Previous works have demonstrated considerable variability in hospital cleanliness in Japan, suggesting that contamination is driven by factors that are currently poorly controlled. We undertook 16S rRNA sequence analysis to study population structures of hospital environmental microbiomes to see which factor(s) impacted contamination. One hundred forty-four samples were collected from surfaces of three hospitals with distinct sizes ("A": >500 beds, "B": 100-500 beds, "C": <100 beds). Sample locations of two ward types (Surgical and Internal) included patient room bed table (multiple) (4BT), patient overbed table (multiple) (4OT), patient room sink (multiple) (4S), patient room bed table (single) (SBT), patient overbed table (single) (SOT), patient room sink (single) (SS), nurse desk (ND), and nurse wagon (NW). Total DNA was extracted from each sample, and the 50 samples that yielded sufficient DNA were used for further 16S rRNA sequencing of hospital microbiome populations with cluster analysis. The number of assigned bacterial OTU populations was significantly decreased in hospital "C" compared to the other hospitals. Cluster analysis of sampling locations revealed that the population structure in almost all locations of hospital "C" and some locations in the other hospitals was very similar and unusually skewed with a family, Enterobacteriaceae. Interestingly, locations included patient area (4OT, 4BT, SBT) and nurse area (ND), with a device (NW) bridging the two and a place (4S and SS) shared between patients or visitors. We demonstrated diversity changes of hospital environmental microbiomes with a skewed population, presumably by medical staff pushing NWs or sinks shared by patients or visitors. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Adhesive blood microsampling systems for steroid measurement via LC-MS/MS in the rat.

PubMed

Heussner, Kirsten; Rauh, Manfred; Cordasic, Nada; Menendez-Castro, Carlos; Huebner, Hanna; Ruebner, Matthias; Schmidt, Marius; Hartner, Andrea; Rascher, Wolfgang; Fahlbusch, Fabian B

2017-04-01

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) allows for the direct analysis of multiple hormones in a single probe with minimal sample volume. Rodent-based animal studies strongly rely on microsampling, such as the dry blood spot (DBS) method. However, DBS suffers the drawback of hematocrit-dependence (non-volumetric). Hence, novel volumetric microsampling techniques were introduced recently, allowing sampling of fixed accurate volumes. We compared these methods for steroid analysis in the rat to improve inter-system comparability. We analyzed steroid levels in blood using the absorptive microsampling devices Whatman® 903 Protein Saver Cards, Noviplex™ Plasma Prep Cards and the Mitra™ Microsampling device and compared the obtained results to the respective EDTA plasma levels. Quantitative steroid analysis was performed via LC-MS/MS. For the determination of the plasma volume factor for each steroid, their levels in pooled blood samples from each human adults and rats (18weeks) were compared and the transferability of these factors was evaluated in a new set of juvenile (21days) and adult (18weeks) rats. Hematocrit was determined concomitantly. Using these approaches, we were unable to apply one single volume factor for each steroid. Instead, plasma volume factors had to be adjusted for the recovery rate of each steroid and device individually. The tested microsampling systems did not allow the use of one single volume factor for adult and juvenile rats based on an unexpectedly strong hematocrit-dependency and other steroid specific (pre-analytic) factors. Our study provides correction factors for LC-MS/MS steroid analysis of volumetric and non-volumetric microsampling systems in comparison to plasma. It argues for thorough analysis of chromatographic effects before the use of novel volumetric systems for steroid analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of x-ray framing cameras for the National Ignition Facility using single photon pulse height analysis.

PubMed

Holder, J P; Benedetti, L R; Bradley, D K

2016-11-01

Single hit pulse height analysis is applied to National Ignition Facility x-ray framing cameras to quantify gain and gain variation in a single micro-channel plate-based instrument. This method allows the separation of gain from detectability in these photon-detecting devices. While pulse heights measured by standard-DC calibration methods follow the expected exponential distribution at the limit of a compound-Poisson process, gain-gated pulse heights follow a more complex distribution that may be approximated as a weighted sum of a few exponentials. We can reproduce this behavior with a simple statistical-sampling model.

Comparison of procedures for correction of matrix interferences in the analysis of soils by ICP-OES with CCD detection

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

Sadler, D.A.; Sun, F.; Littlejohn, D.

1995-12-31

ICP-OES is a useful technique for multi-element analysis of soils. However, as a number of elements are present in relatively high concentrations, matrix interferences can occur and examples have been widely reported. The availability of CCD detectors has increased the opportunities for rapid multi-element, multi-wave-length determination of elemental concentrations in soils and other environmental samples. As the composition of soils from industrial sites can vary considerably, especially when taken from different pit horizons, procedures are required to assess the extent of interferences and correct the effects, on a simultaneous multi-element basis. In single element analysis, plasma operating conditions can sometimesmore » be varied to minimize or even remove multiplicative interferences. In simultaneous multi-element analysis, the scope for this approach may be limited, depending on the spectrochemical characteristics of the emitting analyte species. Matrix matching, by addition of major sample components to the analyte calibrant solutions, can be used to minimize inaccuracies. However, there are also limitations to this procedure, when the sample composition varies significantly. Multiplicative interference effects can also be assessed by a {open_quotes}single standard addition{close_quotes} of each analyte to the sample solution and the information obtained may be used to correct the analyte concentrations determined directly. Each of these approaches has been evaluated to ascertain the best procedure for multi-element analysis of industrial soils by ICP-OES with CCD detection at multiple wavelengths. Standard reference materials and field samples have been analyzed to illustrate the efficacy of each procedure.« less

High-recovery visual identification and single-cell retrieval of circulating tumor cells for genomic analysis using a dual-technology platform integrated with automated immunofluorescence staining.

PubMed

Campton, Daniel E; Ramirez, Arturo B; Nordberg, Joshua J; Drovetto, Nick; Clein, Alisa C; Varshavskaya, Paulina; Friemel, Barry H; Quarre, Steve; Breman, Amy; Dorschner, Michael; Blau, Sibel; Blau, C Anthony; Sabath, Daniel E; Stilwell, Jackie L; Kaldjian, Eric P

2015-05-06

Circulating tumor cells (CTCs) are malignant cells that have migrated from solid cancers into the blood, where they are typically present in rare numbers. There is great interest in using CTCs to monitor response to therapies, to identify clinically actionable biomarkers, and to provide a non-invasive window on the molecular state of a tumor. Here we characterize the performance of the AccuCyte®--CyteFinder® system, a comprehensive, reproducible and highly sensitive platform for collecting, identifying and retrieving individual CTCs from microscopic slides for molecular analysis after automated immunofluorescence staining for epithelial markers. All experiments employed a density-based cell separation apparatus (AccuCyte) to separate nucleated cells from the blood and transfer them to microscopic slides. After staining, the slides were imaged using a digital scanning microscope (CyteFinder). Precisely counted model CTCs (mCTCs) from four cancer cell lines were spiked into whole blood to determine recovery rates. Individual mCTCs were removed from slides using a single-cell retrieval device (CytePicker™) for whole genome amplification and subsequent analysis by PCR and Sanger sequencing, whole exome sequencing, or array-based comparative genomic hybridization. Clinical CTCs were evaluated in blood samples from patients with different cancers in comparison with the CellSearch® system. AccuCyte--CyteFinder presented high-resolution images that allowed identification of mCTCs by morphologic and phenotypic features. Spike-in mCTC recoveries were between 90 and 91%. More than 80% of single-digit spike-in mCTCs were identified and even a single cell in 7.5 mL could be found. Analysis of single SKBR3 mCTCs identified presence of a known TP53 mutation by both PCR and whole exome sequencing, and confirmed the reported karyotype of this cell line. Patient sample CTC counts matched or exceeded CellSearch CTC counts in a small feasibility cohort. The AccuCyte--CyteFinder system is a comprehensive and sensitive platform for identification and characterization of CTCs that has been applied to the assessment of CTCs in cancer patient samples as well as the isolation of single cells for genomic analysis. It thus enables accurate non-invasive monitoring of CTCs and evolving cancer biology for personalized, molecularly-guided cancer treatment.

Characterization of vertical aerosol flows by single particle mass spectrometry for micrometeorological analysis

NASA Astrophysics Data System (ADS)

Gelhausen, Elmar; Hinz, Klaus-Peter; Schmidt, Andres; Spengler, Bernhard

2011-10-01

A single particle mass spectrometer LAMPAS 2 (Laser Mass Analyzer for Particles in the Airborne State) was combined with an ultrasonic anemometer to provide a measurement system for monitoring environmental substance exchange as caused by emission/deposition of aerosol particles. For this study, 681 mass spectra of detected particles were sorted into groups of similarity by a clustering algorithm leading to five classes of different particle types. Each single mass spectrum was correlated to corresponding anemometer data (vertical wind vector and wind speed) in a time-resolved analysis. Due to sampling constraints time-resolution was limited to 36 s, as a result of transition time distributions through the sampling tube. Vertical particle flow (emission/deposition) was determined for all particles based on these data as acquired during a measuring campaign in Giessen, Germany. For a selected particle class a detailed up- and downwards flow consideration was performed to prove the developed approach. Particle flow of that class was dominated by an emission trend as expected. The presented combination of single-particle mass spectrometry and ultrasonic anemometry provides for the possibility to correlate chemical particle data and wind data in a distinct assignment for the description of turbulent particle behavior near earth surface. Results demonstrate the ability to apply the method to real micrometeorological systems, if sampling issues are properly considered for an intended time resolution.

Completely non-destructive elemental analysis of bulky samples by PGAA

NASA Astrophysics Data System (ADS)

Oura, Y.; Nakahara, H.; Sueki, K.; Sato, W.; Saito, A.; Tomizawa, T.; Nishikawa, T.

1999-01-01

NBAA (neutron beam activation analysis), which is a combination of PGAA and INAA by a single neutron irradiation, using an internal monostandard method is proposed as a very unique and promising method for the elemental analysis of voluminous and invaluable archaeological samples which do not allow even a scrape of the surface. It was applied to chinawares, Sueki ware, and bronze mirrors, and proved to be a very effective method for nondestructive analysis of not only major elements but also some minor elements such as boron that help solve archaeological problems of ears and sites of their production.

Technical aspects and recommendations for single-cell qPCR.

PubMed

Ståhlberg, Anders; Kubista, Mikael

2018-02-01

Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tracking Silent Hypersensitivity Reactions to Asparaginase during Leukemia Therapy Using Single-Chip Indirect Plasmonic and Fluorescence Immunosensing.

PubMed

Charbonneau, David M; Breault-Turcot, Julien; Sinnett, Daniel; Krajinovic, Maja; Leclerc, Jean-Marie; Masson, Jean-François; Pelletier, Joelle N

2017-12-22

Microbial asparaginase is an essential component of chemotherapy for the treatment of childhood acute lymphoblastic leukemia (cALL). Silent hypersensitivity reactions to this microbial enzyme need to be monitored accurately during treatment to avoid adverse effects of the drug and its silent inactivation. Here, we present a dual-response anti-asparaginase sensor that combines indirect SPR and fluorescence on a single chip to perform ELISA-type immunosensing, and correlate measurements with classical ELISA. Analysis of serum samples from children undergoing cALL therapy revealed a clear correlation between single-chip indirect SPR/fluorescence immunosensing and ELISA used in clinical settings (R 2 > 0.9). We also report that the portable SPR/fluorescence system had a better sensitivity than classical ELISA to detect antibodies in clinical samples with low antigenicity. This work demonstrates the reliability of dual sensing for monitoring clinically relevant antibody titers in clinical serum samples.

Structural characterization of polysaccharides from bamboo

NASA Astrophysics Data System (ADS)

Kamil, Ruzaimah Nik Mohamad; Yusuf, Nur'aini Raman; Yunus, Normawati M.; Yusup, Suzana

2014-10-01

The alkaline and water soluble polysaccharides were isolate by sequential extractions with distilled water, 60% ethanol containing 1%, 5% and 8% NaOH. The samples were prepared at 60 °C for 3 h from local bamboo. The functional group of the sample were examined using FTIR analysis. The most precipitate obtained is from using 60% ethanol containing 8% NaOH with yield of 2.6%. The former 3 residues isolated by sequential extractions with distilled water, 60% ethanol containing 1% and 5% NaOH are barely visible after filtering with cellulose filter paper. The FTIR result showed that the water-soluble polysaccharides consisted mainly of OH group, CH group, CO indicates the carbohydrate and sugar chain. The sample weight loss was slightly decreased with increasing of temperature.

Evaluation of tools for highly variable gene discovery from single-cell RNA-seq data.

PubMed

Yip, Shun H; Sham, Pak Chung; Wang, Junwen

2018-02-21

Traditional RNA sequencing (RNA-seq) allows the detection of gene expression variations between two or more cell populations through differentially expressed gene (DEG) analysis. However, genes that contribute to cell-to-cell differences are not discoverable with RNA-seq because RNA-seq samples are obtained from a mixture of cells. Single-cell RNA-seq (scRNA-seq) allows the detection of gene expression in each cell. With scRNA-seq, highly variable gene (HVG) discovery allows the detection of genes that contribute strongly to cell-to-cell variation within a homogeneous cell population, such as a population of embryonic stem cells. This analysis is implemented in many software packages. In this study, we compare seven HVG methods from six software packages, including BASiCS, Brennecke, scLVM, scran, scVEGs and Seurat. Our results demonstrate that reproducibility in HVG analysis requires a larger sample size than DEG analysis. Discrepancies between methods and potential issues in these tools are discussed and recommendations are made.

Solid-phase microextraction technology for in vitro and in vivo metabolite analysis

PubMed Central

Zhang, Qihui; Zhou, Liandi; Chen, Hua; Wang, Chong-Zhi; Xia, Zhining; Yuan, Chun-Su

2016-01-01

Analysis of endogenous metabolites in biological samples may lead to the identification of biomarkers in metabolomics studies. To achieve accurate sample analysis, a combined method of continuous quick sampling and extraction is required for online compound detection. Solid-phase microextraction (SPME) integrates sampling, extraction and concentration into a single solvent-free step for chemical analysis. SPME has a number of advantages, including simplicity, high sensitivity and a relatively non-invasive nature. In this article, we reviewed SPME technology in in vitro and in vivo analyses of metabolites after the ingestion of herbal medicines, foods and pharmaceutical agents. The metabolites of microorganisms in dietary supplements and in the gastrointestinal tract will also be examined. As a promising technology in biomedical and pharmaceutical research, SPME and its future applications will depend on advances in analytical technologies and material science. PMID:27695152

DTWscore: differential expression and cell clustering analysis for time-series single-cell RNA-seq data.

PubMed

Wang, Zhuo; Jin, Shuilin; Liu, Guiyou; Zhang, Xiurui; Wang, Nan; Wu, Deliang; Hu, Yang; Zhang, Chiping; Jiang, Qinghua; Xu, Li; Wang, Yadong

2017-05-23

The development of single-cell RNA sequencing has enabled profound discoveries in biology, ranging from the dissection of the composition of complex tissues to the identification of novel cell types and dynamics in some specialized cellular environments. However, the large-scale generation of single-cell RNA-seq (scRNA-seq) data collected at multiple time points remains a challenge to effective measurement gene expression patterns in transcriptome analysis. We present an algorithm based on the Dynamic Time Warping score (DTWscore) combined with time-series data, that enables the detection of gene expression changes across scRNA-seq samples and recovery of potential cell types from complex mixtures of multiple cell types. The DTWscore successfully classify cells of different types with the most highly variable genes from time-series scRNA-seq data. The study was confined to methods that are implemented and available within the R framework. Sample datasets and R packages are available at https://github.com/xiaoxiaoxier/DTWscore .

NM-Scale Anatomy of an Entire Stardust Carrot Track

NASA Technical Reports Server (NTRS)

Nakamura-Messenger, K.; Keller, L. P.; Clemett, S. J.; Messenger, S.

2009-01-01

Comet Wild-2 samples collected by NASA s Stardust mission are extremely complex, heterogeneous, and have experienced wide ranges of alteration during the capture process. There are two major types of track morphologies: "carrot" and "bulbous," that reflect different structural/compositional properties of the impactors. Carrot type tracks are typically produced by compact or single mineral grains which survive essentially intact as a single large terminal particle. Bulbous tracks are likely produced by fine-grained or organic-rich impactors [1]. Owing to their challenging nature and especially high value of Stardust samples, we have invested considerable effort in developing both sample preparation and analytical techniques tailored for Stardust sample analyses. Our report focuses on our systematic disassembly and coordinated analysis of Stardust carrot track #112 from the mm to nm-scale.

Designing single- and multiple-shell sampling schemes for diffusion MRI using spherical code.

PubMed

Cheng, Jian; Shen, Dinggang; Yap, Pew-Thian

2014-01-01

In diffusion MRI (dMRI), determining an appropriate sampling scheme is crucial for acquiring the maximal amount of information for data reconstruction and analysis using the minimal amount of time. For single-shell acquisition, uniform sampling without directional preference is usually favored. To achieve this, a commonly used approach is the Electrostatic Energy Minimization (EEM) method introduced in dMRI by Jones et al. However, the electrostatic energy formulation in EEM is not directly related to the goal of optimal sampling-scheme design, i.e., achieving large angular separation between sampling points. A mathematically more natural approach is to consider the Spherical Code (SC) formulation, which aims to achieve uniform sampling by maximizing the minimal angular difference between sampling points on the unit sphere. Although SC is well studied in the mathematical literature, its current formulation is limited to a single shell and is not applicable to multiple shells. Moreover, SC, or more precisely continuous SC (CSC), currently can only be applied on the continuous unit sphere and hence cannot be used in situations where one or several subsets of sampling points need to be determined from an existing sampling scheme. In this case, discrete SC (DSC) is required. In this paper, we propose novel DSC and CSC methods for designing uniform single-/multi-shell sampling schemes. The DSC and CSC formulations are solved respectively by Mixed Integer Linear Programming (MILP) and a gradient descent approach. A fast greedy incremental solution is also provided for both DSC and CSC. To our knowledge, this is the first work to use SC formulation for designing sampling schemes in dMRI. Experimental results indicate that our methods obtain larger angular separation and better rotational invariance than the generalized EEM (gEEM) method currently used in the Human Connectome Project (HCP).

Thermoelectric properties and thermal stability of Bi-doped PbTe single crystal

NASA Astrophysics Data System (ADS)

Chen, Zhong; Li, Decong; Deng, Shuping; Tang, Yu; Sun, Luqi; Liu, Wenting; Shen, Lanxian; Yang, Peizhi; Deng, Shukang

2018-06-01

In this study, n-type Bi-doped single-crystal PbTe thermoelectric materials were prepared by melting and slow cooling method according to the stoichiometric ratio of Pb:Bi:Te = 1-x:x:1 (x = 0, 0.1, 0.15, 0.2, 0.25). The X-ray diffraction patterns of Pb1-xBixTe samples show that all main diffraction peaks are well matched with the PbTe matrix, which has a face-centered cubic structure with the space group Fm 3 bar m . Electron probe microanalysis reveals that Pb content decreases gradually, and Te content remains invariant basically with the increase of Bi content, indicating that Bi atoms are more likely to replace Pb atoms. Thermal analysis shows that the prepared samples possess relatively high thermal stability. Simultaneously, transmission electron microscopy and selected area electron diffraction pattern indicate that the prepared samples have typical single-crystal structures with good mechanical properties. Moreover, the electrical conductivity of the prepared samples improved significantly compared with that of the pure sample, and the maximum ZT value of 0.84 was obtained at 600 K by the sample with x = 0.2.

Deliberations about a perfect storm - The meaning of justice for food energy water-nexus (FEW-Nexus).

PubMed

Schlör, Holger; Venghaus, Sandra; Fischer, Wiltrud; Märker, Carolin; Hake, Jürgen-Friedrich

2018-08-15

The current global developments have the potential to cause a 'perfect storm' at the core of the Anthropocene: the Food-Energy-Water-Nexus. To discuss the ethical consequences of these developments, i.e., insufficient access to the life sustaining nexus resources, the analysis is focused on Rawls' theory of justice and its implementation in Germany with a special focus on the FEW nexus. Rawls stresses in his theory of justice the prominent meaning of institutions for a fair society to meet societal challenges and to meet the challenge of our time: a stable and just society. Hence, the realization of his ideas in Germany is scrutinized and income tax and value added tax are interpreted in the sense of Douglas North and John Rawls as institutions and formal rules of society. This paper focuses on taxes as the most important institutional incentive to organize and structure the political, social and economic cooperation and analyses how these incentives affect selected German households (all households, singles, single man and woman, and couples) with respect to income and FEW expenditures. The relevant income and usage data sample (Einkommens- und Verbrauchsstichprobe (EVS)) for Germany is used for the analysis of the distribution of income types, FEW expenditures and the revenues of income tax and value added tax, i.e., the main instruments to manage the challenges of the FEW nexus. Therefore two distribution measures have been used: the dispersion of income, taxes and FEW expenditures and their skewness. Five household groups were selected for this analysis: All households, all single households, the single women households, the single men households, as well as the households of couples. The EVS data sample allows the analysis of consequences of the current societal conditions on the various households and thus serves to provide a deeper understanding of the differences between singles and couples but also between single women and men. Copyright © 2018 Elsevier Ltd. All rights reserved.

Over 2,300 phosphorylated peptide identifications with single-shot capillary zone electrophoresis-tandem mass spectrometry in a 100 min separation

PubMed Central

Ludwig, Katelyn R.; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J.; Hummon, Amanda B.

2015-01-01

Ultra-performance liquid chromatography (UPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) is typically employed for phosphoproteome analysis. Alternatively, capillary zone electrophoresis (CZE) - ESI-MS/MS has great potential for phosphoproteome analysis due to the significantly different migration times of phosphorylated and unphosphorylated forms of peptides. In this work, we systematically compared UPLC-MS/MS and CZE-MS/MS for phosphorylated peptide identifications (IDs) using an enriched phosphoproteome from the MCF-10A cell line. When the sample loading amount of UPLC was 10 times higher than that of CZE (2 μg vs. 200 ng), UPLC generated more phosphorylated peptide IDs than CZE (3,313 vs. 1,783). However, when the same sample loading amounts were used for CZE and UPLC (2–200 ng), CZE-MS/MS consistently and significantly outperformed UPLC-MS/MS in terms of phosphorylated peptide and total peptide IDs. This superior performance is most likely due to the higher peptide intensity generated by CZE-MS/MS. More importantly, compared with UPLC data from 2 μg sample, CZE-MS/MS can identify over 500 unique phosphorylated peptides from 200 ng sample, suggesting that CZE and UPLC are complementary for phosphorylated peptide IDs. With further improved loading capacity via a dynamic pH junction method, 2,313 phosphorylated peptides were identified with single-shot CZE-MS/MS in a 100 min analysis. This number of phosphorylated peptide IDs is over one order of magnitude higher than the number of phosphorylated peptide IDs previously reported by single-shot CZE-MS/MS. PMID:26399161

Thermal Decomposition Study on CuInSe2 Single Crystals

NASA Astrophysics Data System (ADS)

Chauhan, Sanjaysinh M.; Chaki, Sunil H.; Deshpande, M. P.; Malek, Tasmira J.; Tailor, J. P.

2018-01-01

The thermal analysis of the chemical vapor transport (CVT)-grown CuInSe2 single crystals was carried out by recording the thermogravimetric, differential thermogravimetric and differential thermal analysis curves. All the three thermo-curves were recorded simultaneously by thermal analyzer in the temperature range of ambient to 1080 K in inert nitrogen atmosphere. The thermo-curves were recorded for four heating rates of 5 K \\cdot min^{-1}, 10 K \\cdot min^{-1}, 15 K \\cdot min^{-1} and 20 K \\cdot min^{-1}. The TG curve analysis showed negligible mass loss in the temperature range of ambient to 600 K, stating the sample material to be thermally stable in this temperature range. Above 601 K to the temperature of 1080 K, the sample showed continuous mass loss. The DTG curves showed two peaks in the temperature range of 601 K to 1080 K. The corresponding DTA showed initial minor exothermic nature followed by endothermic nature up to nearly 750 K and above it showed exothermic nature. The initial exothermic nature is due to absorbed water converting to water vapor, whereas the endothermic nature states the absorption of heat by the sample up to nearly 950 K. Above nearly 950 K the exothermic nature is due to the decomposition of sample material. The absorption of heat in the endothermic region is substantiated by corresponding weight loss in TG. The thermal kinetic parameters of the CVT-grown CuInSe2 single crystals were determined employing the non-mechanistic Kissinger relation. The determined kinetic parameters support the observations of the thermo-curves.

Salmonella testing of pooled pre-enrichment broth cultures for screening multiple food samples.

PubMed

Price, W R; Olsen, R A; Hunter, J E

1972-04-01

A method has been described for testing multiple food samples for Salmonella without loss in sensitivity. The method pools multiple pre-enrichment broth cultures into single enrichment broths. The subsequent stages of the Salmonella analysis are not altered. The method was found applicable to several dry food materials including nonfat dry milk, dried egg albumin, cocoa, cottonseed flour, wheat flour, and shredded coconut. As many as 25 pre-enrichment broth cultures were pooled without apparent loss in the sensitivity of Salmonella detection as compared to individual sample analysis. The procedure offers a simple, yet effective, way to increase sample capacity in the Salmonella testing of foods, particularly where a large proportion of samples ordinarily is negative. It also permits small portions of pre-enrichment broth cultures to be retained for subsequent individual analysis if positive tests are found. Salmonella testing of pooled pre-enrichment broths provides increased consumer protection for a given amount of analytical effort as compared to individual sample analysis.

Dielectrophoresis-Based Sample Handling in General-Purpose Programmable Diagnostic Instruments

PubMed Central

Gascoyne, Peter R. C.; Vykoukal, Jody V.

2009-01-01

As the molecular origins of disease are better understood, the need for affordable, rapid, and automated technologies that enable microscale molecular diagnostics has become apparent. Widespread use of microsystems that perform sample preparation and molecular analysis could ensure that the benefits of new biomedical discoveries are realized by a maximum number of people, even those in environments lacking any infrastructure. While progress has been made in developing miniaturized diagnostic systems, samples are generally processed off-device using labor-intensive and time-consuming traditional sample preparation methods. We present the concept of an integrated programmable general-purpose sample analysis processor (GSAP) architecture where raw samples are routed to separation and analysis functional blocks contained within a single device. Several dielectrophoresis-based methods that could serve as the foundation for building GSAP functional blocks are reviewed including methods for cell and particle sorting, cell focusing, cell ac impedance analysis, cell lysis, and the manipulation of molecules and reagent droplets. PMID:19684877

Crystal Structure Analysis of Electromagnetic Wave Absorber Material BaFe12-xTix/2Znx/2O19Based

NASA Astrophysics Data System (ADS)

Delina, M.; Nenni, N.; Adi, W. A.

2018-04-01

The optimization of BaFe12-xTix/2Znx/2O19 (x=2.2; 2.4; 2.6; 2.8)single phase composition have been performed. The materials were synthesized by solid state reaction method through mechanical milling technique.The materials were made from the mixture of oxide materials, which are BaCO3, Fe2O3, TiO2 and ZnO. The mixture was milled for five hours using a High Energy Milling (HEM), was dried at 100°C in the Oven and then was sintered at 1000°C for five hours in the Furnace. The phase identification of BaFe12-xTix/2Znx/2O19 (x=2.2; 2.4; 2.6; 2.8) were carried out by using a Match Program while the crystal structure analysis were investigated by using a General Structure Analysis System (GSAS) program. The refinement results of x-ray diffraction pattern showed that the sample of x ≤ 2.4 have a BaFe12O19 single phase while the sample of x> 2.4 have two phases, which are BaFe12O19 and ZnFe2O4 phases. The surface morphology of sample and the element of sample were identified through an analysis of Scanning Electron Microscope (SEM) and Energy Dispersive Spectroscopy (EDS) data.

Single-cell forensic short tandem repeat typing within microfluidic droplets.

PubMed

Geng, Tao; Novak, Richard; Mathies, Richard A

2014-01-07

A short tandem repeat (STR) typing method is developed for forensic identification of individual cells. In our strategy, monodisperse 1.5 nL agarose-in-oil droplets are produced with a high frequency using a microfluidic droplet generator. Statistically dilute single cells, along with primer-functionalized microbeads, are randomly compartmentalized in the droplets. Massively parallel single-cell droplet polymerase chain reaction (PCR) is performed to transfer replicas of desired STR targets from the single-cell genomic DNA onto the coencapsulated microbeads. These DNA-conjugated beads are subsequently harvested and reamplified under statistically dilute conditions for conventional capillary electrophoresis (CE) STR fragment size analysis. The 9-plex STR profiles of single cells from both pure and mixed populations of GM09947 and GM09948 human lymphoid cells show that all alleles are correctly called and allelic drop-in/drop-out is not observed. The cell mixture study exhibits a good linear relationship between the observed and input cell ratios in the range of 1:1 to 10:1. Additionally, the STR profile of GM09947 cells could be deduced even in the presence of a high concentration of cell-free contaminating 9948 genomic DNA. Our method will be valuable for the STR analysis of samples containing mixtures of cells/DNA from multiple contributors and for low-concentration samples.

Spatial Genetic Structure and Clonal Diversity in an Alpine Population of Salix herbacea (Salicaceae)

PubMed Central

Reisch, Christoph; Schurm, Sophia; Poschlod, Peter

2007-01-01

Background and Aims Many alpine plant species combine clonal and sexual reproduction to minimize the risks of flowering and seed production in high mountain regions. The spatial genetic structure and diversity of these alpine species is strongly affected by different clonal strategies (phalanx or guerrilla) and the proportion of generative and vegetative reproduction. Methods The clonal structure of the alpine plant species Salix herbacea was investigated in a 3 × 3 m plot of an alpine meadow using microsatellite (simple sequence repeat; SSR) analysis. The data obtained were compared with the results of a random amplified polymorphic DNA (RAPD) analysis. Key Results SSR analysis, based on three loci and 16 alleles, revealed 24 different genotypes and a proportion of distinguishable genotypes of 0·18. Six SSR clones were found consisting of at least five samples, 17 clones consisting of more than two samples and seven single genotypes. Mean clone size comprising at least five samples was 0·96 m2, and spatial autocorrelation analysis showed strong similarity of samples up to 130 cm. RAPD analysis revealed a higher level of clonal diversity but a comparable number of larger clones and a similar spatial structure. Conclusions The spatial genetic structure as well as the occurrence of single genotypes revealed in this study suggests both clonal and sexual propagation and repeated seedling recruitment in established populations of S. herbacea and is thus suggestive of a relaxed phalanx strategy. PMID:17242040

Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline

PubMed Central

Madden, Jeremy T.; Toth, Scott J.; Dettmar, Christopher M.; Newman, Justin A.; Oglesbee, Robert A.; Hedderich, Hartmut G.; Everly, R. Michael; Becker, Michael; Ronau, Judith A.; Buchanan, Susan K.; Cherezov, Vadim; Morrow, Marie E.; Xu, Shenglan; Ferguson, Dale; Makarov, Oleg; Das, Chittaranjan; Fischetti, Robert; Simpson, Garth J.

2013-01-01

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied. PMID:23765294

ELEMENTAL COMPOSITION OF FRESHLY NUCLEATED PARTICLES

EPA Science Inventory

The main objective of this work is to develop a method for real-time sampling and analysis of individual airborne nanoparticles in the 5 - 20 nm diameter range. The size range covered by this method is much smaller than existing single particle methods for chemical analysis. S...

A colorimetric probe based on desensitized ionene-stabilized gold nanoparticles for single-step test for sulfate ions.

PubMed

Arkhipova, Viktoriya V; Apyari, Vladimir V; Dmitrienko, Stanislava G

2015-03-15

Desensitized ionene-stabilized gold nanoparticles have been prepared and applied as a colorimetric probe for the single-step test for sulfate ions at the relatively high concentration level. The approach is based on aggregation of the nanoparticles leading to the change in absorption spectra and color of the solution. These nanoparticles are characterized by the decreased sensitivity due to both electrostatic and steric stabilization, which allows for simple, and rapid direct single-step determination of sulfate at the relatively high concentration level in real water samples without sample pretreatment or dilution. Influence of different factors (the time of interaction, pH, the concentrations of sulfate ions and the nanoparticles) on the aggregation and analytical performance of the procedure was investigated. The method allows for the determination of sulfate ions in the mass range of 0.2-0.4 mg with RSD of 5% from the sample volume of less than 2 mL. It has a sharp dependence of the colorimetric response on the concentration of sulfate, which makes it prospective for indicating deviations of the sulfate concentration regarding some declared value chosen within the above range. The time of the analysis is 2 min. The method was applied to the analysis of mineral water samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Calibrating genomic and allelic coverage bias in single-cell sequencing.

PubMed

Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher

2015-04-16

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.

Calibrating genomic and allelic coverage bias in single-cell sequencing

PubMed Central

Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher

2016-01-01

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913

Microfluidic immunocapture of circulating pancreatic cells using parallel EpCAM and MUC1 capture: characterization, optimization and downstream analysis.

PubMed

Thege, Fredrik I; Lannin, Timothy B; Saha, Trisha N; Tsai, Shannon; Kochman, Michael L; Hollingsworth, Michael A; Rhim, Andrew D; Kirby, Brian J

2014-05-21

We have developed and optimized a microfluidic device platform for the capture and analysis of circulating pancreatic cells (CPCs) and pancreatic circulating tumor cells (CTCs). Our platform uses parallel anti-EpCAM and cancer-specific mucin 1 (MUC1) immunocapture in a silicon microdevice. Using a combination of anti-EpCAM and anti-MUC1 capture in a single device, we are able to achieve efficient capture while extending immunocapture beyond single marker recognition. We also have detected a known oncogenic KRAS mutation in cells spiked in whole blood using immunocapture, RNA extraction, RT-PCR and Sanger sequencing. To allow for downstream single-cell genetic analysis, intact nuclei were released from captured cells by using targeted membrane lysis. We have developed a staining protocol for clinical samples, including standard CTC markers; DAPI, cytokeratin (CK) and CD45, and a novel marker of carcinogenesis in CPCs, mucin 4 (MUC4). We have also demonstrated a semi-automated approach to image analysis and CPC identification, suitable for clinical hypothesis generation. Initial results from immunocapture of a clinical pancreatic cancer patient sample show that parallel capture may capture more of the heterogeneity of the CPC population. With this platform, we aim to develop a diagnostic biomarker for early pancreatic carcinogenesis and patient risk stratification.

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

PubMed

Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

2012-08-15

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

Simultaneous analysis of multiple classes of antimicrobials in environmental water samples using SPE coupled with UHPLC-ESI-MS/MS and isotope dilution.

PubMed

Tran, Ngoc Han; Chen, Hongjie; Do, Thanh Van; Reinhard, Martin; Ngo, Huu Hao; He, Yiliang; Gin, Karina Yew-Hoong

2016-10-01

A robust and sensitive analytical method was developed for the simultaneous analysis of 21 target antimicrobials in different environmental water samples. Both single SPE and tandem SPE cartridge systems were investigated to simultaneously extract multiple classes of antimicrobials. Experimental results showed that good extraction efficiencies (84.5-105.6%) were observed for the vast majority of the target analytes when extraction was performed using the tandem SPE cartridge (SB+HR-X) system under an extraction pH of 3.0. HPLC-MS/MS parameters were optimized for simultaneous analysis of all the target analytes in a single injection. Quantification of target antimicrobials in water samples was accomplished using 15 isotopically labeled internal standards (ILISs), which allowed the efficient compensation of the losses of target analytes during sample preparation and correction of matrix effects during UHPLC-MS/MS as well as instrument fluctuations in MS/MS signal intensity. Method quantification limit (MQL) for most target analytes based on SPE was below 5ng/L for surface waters, 10ng/L for treated wastewater effluents, and 15ng/L for raw wastewater. The method was successfully applied to detect and quantify the occurrence of the target analytes in raw influent, treated effluent and surface water samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression and mutational analysis of Cip/Kip family in early glottic cancer.

PubMed

Kim, D-K; Lee, J H; Lee, O J; Park, C H

2015-02-01

Genetic alteration of cyclin-dependent kinase inhibitors has been associated with carcinogenesis mechanisms in various organs. This study aimed to evaluate the expression and mutational analysis of Cip/Kip family cyclin-dependent kinase inhibitors (p21CIP1/WAF1, p27KIP1 and p57KIP2) in early glottic cancer. Expressions of Cip/Kip family and p53 were determined by quantitative reverse transcription polymerase chain reaction and densitometry. For the analysis of p21 inactivation, sequence alteration was assessed using single-strand conformational polymorphism polymerase chain reaction. Additionally, the inactivation mechanism of p27 and p57 were investigated using DNA methylation analysis. Reduced expression of p27 and p57 were detected in all samples, whereas the expression of p21 was incompletely down-regulated in 6 of 11 samples. Additionally, single-strand conformational polymorphism polymerase chain reaction analysis showed the p53 mutation at exon 6. Methylation of p27 and p57 was detected by DNA methylation assay. Our results suggest that the Cip/Kip family may have a role as a molecular mechanism of carcinogenesis in early glottic cancer.

Multi-analysis determination of tropane alkaloids in cereals and solanaceaes seeds by liquid chromatography coupled to single stage Exactive-Orbitrap.

PubMed

Marín-Sáez, Jesús; Romero-González, Roberto; Garrido Frenich, Antonia

2017-10-06

Tropane alkaloids are a wide group of substances that comprises more than 200 compounds occurring especially in the Solanaceae family. The main aim of this study is the development of a method for the analysis of the principal tropane alkaloids as atropine, scopolamine, anisodamine, tropane, tropine, littorine, homatropine, apoatropine, aposcopolamine, scopoline, tropinone, physoperuvine, pseudotropine and cuscohygrine in cereals and related matrices. For that, a simple solid-liquid extraction was optimized and a liquid chromatographic method coupled to a single stage Exactive-Orbitrap was developed. The method was validated obtaining recoveries in the range of 60-109% (except for some compounds in soy), precision values (expressed as relative standard deviation) lower than 20% and detection and quantification limits equal to or lower than 2 and 3μg/kg respectively. Finally, the method was applied to the analysis of different types of samples as buckwheat, linseed, soy and millet, obtaining positives for anisodamine, scopolamine, atropine, littorine and tropinone in a millet flour sample above the quantification limits, whereas atropine and scopolamine were detected in a buckwheat sample, below the quantification limit. Contaminated samples with Solanaceaes seeds (Datura Stramonium and Brugmansia Arborea) were also analysed, detecting concentrations up to 693μg/kg (scopolamine) for contaminated samples with Brugmansia seeds and 1847μg/kg (atropine) when samples were contaminated with Stramonium seeds. Copyright © 2017 Elsevier B.V. All rights reserved.

Diagnostic Accuracy and Cost-Effectiveness of Alternative Methods for Detection of Soil-Transmitted Helminths in a Post-Treatment Setting in Western Kenya

PubMed Central

Kepha, Stella; Kihara, Jimmy H.; Njenga, Sammy M.; Pullan, Rachel L.; Brooker, Simon J.

2014-01-01

Objectives This study evaluates the diagnostic accuracy and cost-effectiveness of the Kato-Katz and Mini-FLOTAC methods for detection of soil-transmitted helminths (STH) in a post-treatment setting in western Kenya. A cost analysis also explores the cost implications of collecting samples during school surveys when compared to household surveys. Methods Stool samples were collected from children (n = 652) attending 18 schools in Bungoma County and diagnosed by the Kato-Katz and Mini-FLOTAC coprological methods. Sensitivity and additional diagnostic performance measures were analyzed using Bayesian latent class modeling. Financial and economic costs were calculated for all survey and diagnostic activities, and cost per child tested, cost per case detected and cost per STH infection correctly classified were estimated. A sensitivity analysis was conducted to assess the impact of various survey parameters on cost estimates. Results Both diagnostic methods exhibited comparable sensitivity for detection of any STH species over single and consecutive day sampling: 52.0% for single day Kato-Katz; 49.1% for single-day Mini-FLOTAC; 76.9% for consecutive day Kato-Katz; and 74.1% for consecutive day Mini-FLOTAC. Diagnostic performance did not differ significantly between methods for the different STH species. Use of Kato-Katz with school-based sampling was the lowest cost scenario for cost per child tested ($10.14) and cost per case correctly classified ($12.84). Cost per case detected was lowest for Kato-Katz used in community-based sampling ($128.24). Sensitivity analysis revealed the cost of case detection for any STH decreased non-linearly as prevalence rates increased and was influenced by the number of samples collected. Conclusions The Kato-Katz method was comparable in diagnostic sensitivity to the Mini-FLOTAC method, but afforded greater cost-effectiveness. Future work is required to evaluate the cost-effectiveness of STH surveillance in different settings. PMID:24810593

Diagnostic accuracy and cost-effectiveness of alternative methods for detection of soil-transmitted helminths in a post-treatment setting in western Kenya.

PubMed

Assefa, Liya M; Crellen, Thomas; Kepha, Stella; Kihara, Jimmy H; Njenga, Sammy M; Pullan, Rachel L; Brooker, Simon J

2014-05-01

This study evaluates the diagnostic accuracy and cost-effectiveness of the Kato-Katz and Mini-FLOTAC methods for detection of soil-transmitted helminths (STH) in a post-treatment setting in western Kenya. A cost analysis also explores the cost implications of collecting samples during school surveys when compared to household surveys. Stool samples were collected from children (n = 652) attending 18 schools in Bungoma County and diagnosed by the Kato-Katz and Mini-FLOTAC coprological methods. Sensitivity and additional diagnostic performance measures were analyzed using Bayesian latent class modeling. Financial and economic costs were calculated for all survey and diagnostic activities, and cost per child tested, cost per case detected and cost per STH infection correctly classified were estimated. A sensitivity analysis was conducted to assess the impact of various survey parameters on cost estimates. Both diagnostic methods exhibited comparable sensitivity for detection of any STH species over single and consecutive day sampling: 52.0% for single day Kato-Katz; 49.1% for single-day Mini-FLOTAC; 76.9% for consecutive day Kato-Katz; and 74.1% for consecutive day Mini-FLOTAC. Diagnostic performance did not differ significantly between methods for the different STH species. Use of Kato-Katz with school-based sampling was the lowest cost scenario for cost per child tested ($10.14) and cost per case correctly classified ($12.84). Cost per case detected was lowest for Kato-Katz used in community-based sampling ($128.24). Sensitivity analysis revealed the cost of case detection for any STH decreased non-linearly as prevalence rates increased and was influenced by the number of samples collected. The Kato-Katz method was comparable in diagnostic sensitivity to the Mini-FLOTAC method, but afforded greater cost-effectiveness. Future work is required to evaluate the cost-effectiveness of STH surveillance in different settings.

Experiments on Nucleation in Different Flow Regimes

NASA Technical Reports Server (NTRS)

Bayuzick, R. J.; Hofmeister, W. H.; Morton, C. M.; Robinson, M. B.

1999-01-01

The vast majority of metallic engineering materials are solidified from the liquid phase. Understanding the solidification process is essential to control microstructure, which in turn, determines the properties of materials. The genesis of solidification is nucleation, where the first stable solid forms from the liquid phase. Nucleation kinetics determine the degree of undercooling and phase selection. As such, it is important to understand nucleation phenomena in order to control solidification or glass formation in metals and alloys. Early experiments in nucleation kinetics were accomplished by droplet dispersion methods. Dilatometry was used by Turnbull and others, and more recently differential thermal analysis and differential scanning calorimetry have been used for kinetic studies. These techniques have enjoyed success; however, there are difficulties with these experiments. Since materials are dispersed in a medium, the character of the emulsion/metal interface affects the nucleation behavior. Statistics are derived from the large number of particles observed in a single experiment, but dispersions have a finite size distribution which adds to the uncertainty of the kinetic determinations. Even though temperature can be controlled quite well before the onset of nucleation, the release of the latent heat of fusion during nucleation of particles complicates the assumption of isothermality during these experiments. Containerless processing has enabled another approach to the study of nucleation kinetics. With levitation techniques it is possible to undercool one sample to nucleation repeatedly in a controlled manner, such that the statistics of the nucleation process can be derived from multiple experiments on a single sample. The authors have fully developed the analysis of nucleation experiments on single samples following the suggestions of Skripov. The advantage of these experiments is that the samples are directly observable. The nucleation temperature can be measured by noncontact optical pyrometry, the mass of the sample is known, and post processing analysis can be conducted on the sample. The disadvantages are that temperature measurement must have exceptionally high precision, and it is not possible to isolate specific heterogeneous sites as in droplet dispersions.

Diffraction data of core-shell nanoparticles from an X-ray free electron laser

DOE PAGES

Li, Xuanxuan; Chiu, Chun -Ya; Wang, Hsiang -Ju; ...

2017-04-11

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Furthermore, scattering patterns resulting from single particles were selected and compiledmore » into a dataset which can be valuable for algorithm developments in single particle scattering research.« less

Resonance Raman and UV-visible spectroscopy of black dyes on textiles.

PubMed

Abbott, Laurence C; Batchelor, Stephen N; Smith, John R Lindsay; Moore, John N

2010-10-10

Resonance Raman and UV-visible diffuse reflectance spectra were recorded from samples of cotton, viscose, polyester, nylon, and acrylic textile swatches dyed black with one of seven single dyes, a mixture of two dyes, or one of seven mixtures of three dyes. The samples generally gave characteristic Raman spectra of the dyes, demonstrating that the technique is applicable for the forensic analysis of dyed black textiles. Survey studies of the widely used dye Reactive Black 5 show that essentially the same Raman spectrum is obtained on bulk sampling from the dye in solution, on viscose, on cotton at different uptakes, and on microscope sampling from the dye in cotton threads and single fibres. The effects of laser irradiation on the Raman bands and emission backgrounds from textile samples with and without dye are also reported. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

High sensitivity Troponin T: an audit of implementation of its protocol in a district general hospital.

PubMed

Kalim, Shahid; Nazir, Shaista; Khan, Zia Ullah

2013-01-01

Protocols based on newer high sensitivity Troponin T (hsTropT) assays can rule in a suspected Acute Myocardial Infarction (AMI) as early as 3 hours. We conducted this study to audit adherence to our Trust's newly introduced AMI diagnostic protocol based on paired hsTropT testing at 0 and 3 hours. We retrospectively reviewed data of all patients who had hsTropT test done between 1st and 7th May 2012. Patient's demographics, utility of single or paired samples, time interval between paired samples, patient's presenting symptoms and ECG findings were noted and their means, medians, Standard deviations and proportions were calculated. A total of 66 patients had hsTropT test done during this period. Mean age was 63.30 +/- 17.46 years and 38 (57.57%) were males. Twenty-four (36.36%) patients had only single, rather than protocol recommended paired hsTropT samples, taken. Among the 42 (63.63%) patients with paired samples, the mean time interval was found to be 4.41 +/- 5.7 hours. Contrary to the recommendations, 15 (22.73%) had a very long whereas 2 (3.03%) had a very short time interval between two samples. A subgroup analysis of patients with single samples, found only 2 (3.03%) patient with ST-segment elevation, appropriate for single testing. Our study confirmed that in a large number of patients the protocol for paired sampling or a recommended time interval of 3 hours between 2 samples was not being followed.

Thermoelectric Properties of Sr-Filled Ge-Based Type I Single-Crystal Clathrate Grown by Sn-Flux Method

NASA Astrophysics Data System (ADS)

Deng, Shuping; Liu, Hongxia; Li, Decong; Wang, Jinsong; Cheng, Feng; Shen, Lanxian; Deng, Shukang

2017-05-01

Single-crystal samples of Sr-filled Ge-based type I clathrate have been prepared by the Sn-flux method, and their thermoelectric properties investigated. The obtained samples exhibited n-type conduction with carrier concentration varying from 2.8 × 1019/cm3 to 6.8 × 1019/cm3 as the carrier mobility changed from 23.9 cm2/V-s to 15.1 cm2/V-s at room temperature. Structural analysis indicated that all samples were type I clathrate in space group pm\\bar{it{3}}n . The total content of group IV (Ge + Sn) atoms in the crystalline structure increased with increasing x value (where x defines the atomic ratio of starting elements, Sr:Ga:Ge:Sn = 8:16: x:20), reaching a maximum value of 31.76 at.% for the sample with x = 30; consequently, the lattice parameters increased. The melting points for all samples were approximately 1012 K, being considerably lower than that of single-crystal Sr8Ga16Ge30 prepared by other methods. The electrical conductivity increased while the absolute value of α increased gradually with increasing temperature; the maximum value of α reached 193 μV/K at 750 K for the sample with x = 24. The sample with x = 30 exhibited lower lattice thermal conductivity of 0.80 W/m-K. As a result, among all the Sn-flux samples, single-crystal Sr7.92Ga15.04Sn0.35Ge30.69 had the largest ZT value of 1.0 at about 750 K.

Analysis of shockless dynamic compression data on solids to multi-megabar pressures: Application to tantalum

DOE PAGES

Davis, Jean -Paul; Brown, Justin L.; Knudson, Marcus D.; ...

2014-11-26

In this research, magnetically-driven, planar shockless-compression experiments to multi-megabar pressures were performed on tantalum samples using a stripline target geometry. Free-surface velocity waveforms were measured in 15 cases; nine of these in a dual-sample configuration with two samples of different thicknesses on opposing electrodes, and six in a single-sample configuration with a bare electrode opposite the sample. Details are given on the application of inverse Lagrangian analysis (ILA) to these data, including potential sources of error. The most significant source of systematic error, particularly for single-sample experiments, was found to arise from the pulse-shape dependent free-surface reflected wave interactions withmore » the deviatoric-stress response of tantalum. This could cause local, possibly temporary, unloading of material from a ramp compressed state, and thus multi-value response in wave speed that invalidates the free-surface to in-material velocity mapping step of ILA. By averaging all 15 data sets, a final result for the principal quasi-isentrope of tantalum in stress-strain was obtained to a peak longitudinal stress of 330 GPa with conservative uncertainty bounds of ±4.5% in stress. The result agrees well with a tabular equation of state developed at Los Alamos National Laboratory.« less

Conic Sector Analysis of Hybrid Control Systems. Ph.D. Thesis

NASA Technical Reports Server (NTRS)

Thompson, P. M.

1982-01-01

A hybrid control system contains an analog plant and a hybrid (or sampled-data) compensator. In this thesis a new conic sector is determined which is constructive and can be used to: (1) determine closed loop stability, (2) analyze robustness with respect to modelling uncertainties, (3) analyze steady state response to commands, and (4) select the sample rate. The use of conic sectors allows the designer to treat hybrid control systems as though they were analog control systems. The center of the conic sector can be used as a rigorous linear time invariant approximation of the hybrid control system, and the radius places a bound on the errors of this approximation. The hybrid feedback system can be multivariable, and the sampler is assumed to be synchronous. Algorithms to compute the conic sector are presented. Several examples demonstrate how the conic sector analysis techniques are applied. Extensions to single loop multirate hybrid feedback systems are presented. Further extensions are proposed for multiloop multirate hybrid feedback system and for single rate systems with asynchronous sampling.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Gene Expression Profiling of Multiple Leiomyomata Uteri and Matched Normal Tissue from a Single Patient

PubMed Central

Dimitrova, Irina K.; Richer, Jennifer K.; Rudolph, Michael C.; Spoelstra, Nicole S.; Reno, Elaine M.; Medina, Theresa M.; Bradford, Andrew P.

2009-01-01

Objective To identify differentially expressed genes between fibroid and adjacent normal myometrium in an identical hormonal and genetic background. Design Array analysis of 3 leiomyomata and matched adjacent normal myometrium in a single patient. Setting University of Colorado Hospital. Patient(s) A single female undergoing medically indicated hysterectomy for symptomatic fibroids. Interventions(s) mRNA isolation and microarray analysis, reverse-transcriptase polymerase chain reaction, western blotting and immunohistochemistry. Main Outcome Measure(s) Changes in mRNA and protein levels in leiomyomata and matched normal myometrium. Result(s) Expression of 197 genes was increased and 619 decreased, significantly by at least 2 fold, in leiomyomata relative to normal myometrium. Expression profiles between tumors were similar and normal myometrial samples showed minimal variation. Changes in, and variation of, expression of selected genes were confirmed in additional normal and leiomyoma samples from multiple patients. Conclusion(s) Analysis of multiple tumors from a single patient confirmed changes in expression of genes described in previous, apparently disparate, studies and identified novel targets. Gene expression profiles in leiomyomata are consistent with increased activation of mitogenic pathways and inhibition of apoptosis. Down-regulation of genes implicated in invasion and metastasis, of cancers, was observed in fibroids. This expression pattern may underlie the benign nature of uterine leiomyomata and may aid in the differential diagnosis of leiomyosarcoma. PMID:18672237

Determining Sample Sizes for Precise Contrast Analysis with Heterogeneous Variances

ERIC Educational Resources Information Center

Jan, Show-Li; Shieh, Gwowen

2014-01-01

The analysis of variance (ANOVA) is one of the most frequently used statistical analyses in practical applications. Accordingly, the single and multiple comparison procedures are frequently applied to assess the differences among mean effects. However, the underlying assumption of homogeneous variances may not always be tenable. This study…

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

Gundlach-Graham, Alexander W.; Dennis, Elise; Ray, Steven J.

An inductively coupled plasma distance-of-flight mass spectrometer (ICP-DOFMS) has been coupled with laser-ablation (LA) sample introduction for the elemental analysis of solids. ICP-DOFMS is well suited for the analysis of laser-generated aerosols because it offers both high-speed mass analysis and simultaneous multi-elemental detection. Here, we evaluate the analytical performance of the LA-ICP-DOFMS instrument, equipped with a microchannel plate-based imaging detector, for the measurement of steady-state LA signals, as well as transient signals produced from single LA events. Steady-state detection limits are 1 mg g1, and absolute single-pulse LA detection limits are 200 fg for uranium; the system is shown capablemore » of performing time-resolved single-pulse LA analysis. By leveraging the benefits of simultaneous multi-elemental detection, we also attain a good shot-to-shot reproducibility of 6% relative standard deviation (RSD) and isotope-ratio precision of 0.3% RSD with a 10 s integration time.« less

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

PubMed

Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

2014-03-18

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

Bioinformatics approaches to single-cell analysis in developmental biology.

PubMed

Yalcin, Dicle; Hakguder, Zeynep M; Otu, Hasan H

2016-03-01

Individual cells within the same population show various degrees of heterogeneity, which may be better handled with single-cell analysis to address biological and clinical questions. Single-cell analysis is especially important in developmental biology as subtle spatial and temporal differences in cells have significant associations with cell fate decisions during differentiation and with the description of a particular state of a cell exhibiting an aberrant phenotype. Biotechnological advances, especially in the area of microfluidics, have led to a robust, massively parallel and multi-dimensional capturing, sorting, and lysis of single-cells and amplification of related macromolecules, which have enabled the use of imaging and omics techniques on single cells. There have been improvements in computational single-cell image analysis in developmental biology regarding feature extraction, segmentation, image enhancement and machine learning, handling limitations of optical resolution to gain new perspectives from the raw microscopy images. Omics approaches, such as transcriptomics, genomics and epigenomics, targeting gene and small RNA expression, single nucleotide and structural variations and methylation and histone modifications, rely heavily on high-throughput sequencing technologies. Although there are well-established bioinformatics methods for analysis of sequence data, there are limited bioinformatics approaches which address experimental design, sample size considerations, amplification bias, normalization, differential expression, coverage, clustering and classification issues, specifically applied at the single-cell level. In this review, we summarize biological and technological advancements, discuss challenges faced in the aforementioned data acquisition and analysis issues and present future prospects for application of single-cell analyses to developmental biology. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.

PubMed

Matsunaga, Hiroko; Goto, Mari; Arikawa, Koji; Shirai, Masataka; Tsunoda, Hiroyuki; Huang, Huan; Kambara, Hideki

2015-02-15

Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process. By adding the washing steps, the next steps can be carried out under the optimal conditions without losing cDNAs. Error sources were carefully evaluated to conclude that the first several steps were the key steps. It is demonstrated that bead-seq is superior to the conventional methods for single-cell gene expression analyses in terms of reproducibility, quantitative accuracy, and biases caused during sample preparation and sequencing processes. Copyright © 2014 Elsevier Inc. All rights reserved.

Nanopipettes: probes for local sample analysis.

PubMed

Saha-Shah, Anumita; Weber, Anna E; Karty, Jonathan A; Ray, Steven J; Hieftje, Gary M; Baker, Lane A

2015-06-01

Nanopipettes (pipettes with diameters <1 μm) were explored as pressure-driven fluid manipulation tools for sampling nanoliter volumes of fluids. The fundamental behavior of fluids confined in the narrow channels of the nanopipette shank was studied to optimize sampling volume and probe geometry. This method was utilized to collect nanoliter volumes (<10 nL) of sample from single Allium cepa cells and live Drosophila melanogaster first instar larvae. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was utilized to characterize the collected sample. The use of nanopipettes for surface sampling of mouse brain tissue sections was also explored. Lipid analyses were performed on mouse brain tissues with spatial resolution of sampling as small as 50 μm. Nanopipettes were shown to be a versatile tool that will find further application in studies of sample heterogeneity and population analysis for a wide range of samples.

Single cell-based analysis of torenia petal pigments by a combination of ArF excimer laser micro sampling and nano-high performance liquid chromatography (HPLC)-mass spectrometry.

PubMed

Kajiyama, Shin'ichiro; Harada, Kazuo; Fukusaki, Eiichiro; Kobayashi, Akio

2006-12-01

The molecular constituents of the petal pigments of the Torenia plant (Torenia hybrida) were analyzed on a single-cell basis by a combination of newly developed laser-microsampling and nano-flow liquid chromatography-electro spray ionization mass spectrometry (LC-ESIMS) techniques. Our method should provide a facile method for obtaining precise metabolic profiles of each cell in a single plant tissue.

The Determination of Sediment Polycyclic Aromatic Hydrocarbon (PAH) Bioavailability using Direct Pore Water Analysis by Solid-Phase Microextraction (SPME)

DTIC Science & Technology

2010-08-01

available). It is assumed after this method is formally published that various standard vendors will offer other sources than the current single standard... single isomer. D Alkyl PAHs used to determine the SPME-GC/MS relative response factors including alkyl naphthalenes (1-methyl-, 2-methyl-, 1,2...Flag all compound results in the sample which were estimated above the upper calibration level with an “E” qualifier. 15. Precision and Bias 15.1 Single

Czochralski growth of LaPd2Al2 single crystals

NASA Astrophysics Data System (ADS)

Doležal, P.; Rudajevová, A.; Vlášková, K.; Kriegner, D.; Václavová, K.; Prchal, J.; Javorský, P.

2017-10-01

The present study is focused on the preparation of single crystalline LaPd2Al2 by the Czochralski method. Differential scanning calorimetry (DSC) and energy dispersive X-ray spectroscopy (EDX) analyses reveal that LaPd2Al2 is an incongruently melting phase which causes difficulties for the preparation of single crystalline LaPd2Al2 by the Czochralski method. Therefore several non-stoichiometric polycrystalline samples were studied for its preparation. Finally the successful growth of LaPd2Al2 without foreign phases has been achieved by using a non-stoichiometric precursor with atomic composition 22:39:39 (La:Pd:Al). X-ray powder diffraction, EDX analysis and DSC were used for the characterisation. A single crystalline sample was separated from the ingot prepared by the Czochralski method using the non-stoichiometric precursor. The presented procedure for the preparation of pure single phase LaPd2Al2 could be modified for other incongruently melting phases.

Correcting for surface topography in X-ray fluorescence imaging

PubMed Central

Geil, E. C.; Thorne, R. E.

2014-01-01

Samples with non-planar surfaces present challenges for X-ray fluorescence imaging analysis. Here, approximations are derived to describe the modulation of fluorescence signals by surface angles and topography, and suggestions are made for reducing this effect. A correction procedure is developed that is effective for trace element analysis of samples having a uniform matrix, and requires only a fluorescence map from a single detector. This procedure is applied to fluorescence maps from an incised gypsum tablet. PMID:25343805

Analysis of single ion channel data incorporating time-interval omission and sampling

PubMed Central

The, Yu-Kai; Timmer, Jens

2005-01-01

Hidden Markov models are widely used to describe single channel currents from patch-clamp experiments. The inevitable anti-aliasing filter limits the time resolution of the measurements and therefore the standard hidden Markov model is not adequate anymore. The notion of time-interval omission has been introduced where brief events are not detected. The developed, exact solutions to this problem do not take into account that the measured intervals are limited by the sampling time. In this case the dead-time that specifies the minimal detectable interval length is not defined unambiguously. We show that a wrong choice of the dead-time leads to considerably biased estimates and present the appropriate equations to describe sampled data. PMID:16849220

Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples.

PubMed

Lam, Maggie P Y; Lau, Edward; Siu, S O; Ng, Dominic C M; Kong, Ricky P W; Chiu, Philip C N; Yeung, William S B; Lo, Clive; Chu, Ivan K

2011-11-01

In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High throughput analysis of samples in flowing liquid

DOEpatents

Ambrose, W. Patrick; Grace, W. Kevin; Goodwin, Peter M.; Jett, James H.; Orden, Alan Van; Keller, Richard A.

2001-01-01

Apparatus and method enable imaging multiple fluorescent sample particles in a single flow channel. A flow channel defines a flow direction for samples in a flow stream and has a viewing plane perpendicular to the flow direction. A laser beam is formed as a ribbon having a width effective to cover the viewing plane. Imaging optics are arranged to view the viewing plane to form an image of the fluorescent sample particles in the flow stream, and a camera records the image formed by the imaging optics.

Single cell transcriptomic analysis of prostate cancer cells.

PubMed

Welty, Christopher J; Coleman, Ilsa; Coleman, Roger; Lakely, Bryce; Xia, Jing; Chen, Shu; Gulati, Roman; Larson, Sandy R; Lange, Paul H; Montgomery, Bruce; Nelson, Peter S; Vessella, Robert L; Morrissey, Colm

2013-02-16

The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). To determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell, we clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, two groups of 10 individual DTC, each isolated from bone marrow of 2 patients with metastatic PCa were obtained. RNA was amplified using the WT-Ovation™ One-Direct Amplification System. The amplified material was hybridized on a 44K Whole Human Gene Expression Microarray. A high stringency threshold, a mean Alexa Fluor® 3 signal intensity above 300, was used for gene detection. Relative expression levels were validated for select genes using real-time PCR (RT-qPCR). Using this approach, 22,410, 20,423, and 17,009 probes were positive on the arrays from 10-cell pools, 5-cell pools, and single-cells, respectively. The sensitivity and specificity of gene detection on the single-cell analyses were 0.739 and 0.972 respectively when compared to 10-cell pools, and 0.814 and 0.979 respectively when compared to 5-cell pools, demonstrating a low false positive rate. Among 10,000 randomly selected pairs of genes, the Pearson correlation coefficient was 0.875 between the single-cell and 5-cell pools and 0.783 between the single-cell and 10-cell pools. As expected, abundant transcripts in the 5- and 10-cell samples were detected by RT-qPCR in the single-cell isolates, while lower abundance messages were not. Using the same stringency, 16,039 probes were positive on the patient single-cell arrays. Cluster analysis showed that all 10 DTC grouped together within each patient. A transcriptomic profile can be reliably obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled-cell samples, however this method can be used to reliably obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa.

Single sample resolution of rare microbial dark matter in a marine invertebrate metagenome

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

Miller, Ian J.; Weyna, Theodore R.; Fong, Stephen S.

Direct, untargeted sequencing of environmental samples (metagenomics) and de novo genome assembly enable the study of uncultured and phylogenetically divergent organisms. However, separating individual genomes from a mixed community has often relied on the differential-coverage analysis of multiple, deeply sequenced samples. In the metagenomic investigation of the marine bryozoan Bugula neritina, we uncovered seven bacterial genomes associated with a single B. neritina individual that appeared to be transient associates, two of which were unique to one individual and undetectable using certain “universal” 16S rRNA primers and probes. We recovered high quality genome assemblies for several rare instances of “microbial darkmore » matter,” or phylogenetically divergent bacteria lacking genomes in reference databases, from a single tissue sample that was not subjected to any physical or chemical pre-treatment. One of these rare, divergent organisms has a small (593 kbp), poorly annotated genome with low GC content (20.9%) and a 16S rRNA gene with just 65% sequence similarity to the closest reference sequence. Lastly, our findings illustrate the importance of sampling strategy and de novo assembly of metagenomic reads to understand the extent and function of bacterial biodiversity.« less

Single sample resolution of rare microbial dark matter in a marine invertebrate metagenome

DOE PAGES

Miller, Ian J.; Weyna, Theodore R.; Fong, Stephen S.; ...

2016-09-29

Direct, untargeted sequencing of environmental samples (metagenomics) and de novo genome assembly enable the study of uncultured and phylogenetically divergent organisms. However, separating individual genomes from a mixed community has often relied on the differential-coverage analysis of multiple, deeply sequenced samples. In the metagenomic investigation of the marine bryozoan Bugula neritina, we uncovered seven bacterial genomes associated with a single B. neritina individual that appeared to be transient associates, two of which were unique to one individual and undetectable using certain “universal” 16S rRNA primers and probes. We recovered high quality genome assemblies for several rare instances of “microbial darkmore » matter,” or phylogenetically divergent bacteria lacking genomes in reference databases, from a single tissue sample that was not subjected to any physical or chemical pre-treatment. One of these rare, divergent organisms has a small (593 kbp), poorly annotated genome with low GC content (20.9%) and a 16S rRNA gene with just 65% sequence similarity to the closest reference sequence. Lastly, our findings illustrate the importance of sampling strategy and de novo assembly of metagenomic reads to understand the extent and function of bacterial biodiversity.« less

Contaminant characterization on hair and fiber surfaces using imaging TOF-SIMS

NASA Astrophysics Data System (ADS)

Groenewold, Gary S.; Gresham, Garold L.; Gianotto, Anita K.; Avci, Recep

1999-02-01

Imaging time-of-flight secondary ion mass spectrometry (SIMS) was used to evaluate the detection of contaminant chemicals on the surfaces of single synthetic textile and canine hair fibers. The results of the study showed that a variety of chemical classes can be detected. Both cocaine and heroin could be easily observed as intact protonated molecules ([M + H]+) in the cation spectra acquired from textile fibers. Two organophosphates were evaluated: malathion, which is a common pesticide, and pinacolyl methyl phosphonic acid (PMPA), which is the principal degradation product of the nerve agent soman (a close relative of sarin). Malathion could be observed as (CH3O)2P(equalsS)S-, which is formed by thiophosphate cleavage of the intact malathion. PMPA is observed as the conjugate base ([PMPA - H]-). Surfactant chemicals found in hair care products were successfully detected on single hair fibers. Specifically, alkyl sulfates, ethoxylated alkyl sulfates, silicones, and alkylammonium compounds could be readily identified in spectra acquired from single hair fiber samples exposed to shampoo and/or conditioner. Generally, the results of the study show that imaging SIMS is applicable to single fiber analysis, for a range of adsorbed compound types. The forensic application of this instrumental approach has not been widely recognized. However, the ability of the technique to acquire specific chemical information from trace samples clearly points to applications where the need for chemical analysis is great, but the amount of sample is limited.

Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics.

PubMed

Farlik, Matthias; Sheffield, Nathan C; Nuzzo, Angelo; Datlinger, Paul; Schönegger, Andreas; Klughammer, Johanna; Bock, Christoph

2015-03-03

Methods for single-cell genome and transcriptome sequencing have contributed to our understanding of cellular heterogeneity, whereas methods for single-cell epigenomics are much less established. Here, we describe a whole-genome bisulfite sequencing (WGBS) assay that enables DNA methylation mapping in very small cell populations (μWGBS) and single cells (scWGBS). Our assay is optimized for profiling many samples at low coverage, and we describe a bioinformatic method that analyzes collections of single-cell methylomes to infer cell-state dynamics. Using these technological advances, we studied epigenomic cell-state dynamics in three in vitro models of cellular differentiation and pluripotency, where we observed characteristic patterns of epigenome remodeling and cell-to-cell heterogeneity. The described method enables single-cell analysis of DNA methylation in a broad range of biological systems, including embryonic development, stem cell differentiation, and cancer. It can also be used to establish composite methylomes that account for cell-to-cell heterogeneity in complex tissue samples. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Nuclear reaction analysis of Ge ion-implanted ZnO bulk single crystals: The evaluation of the displacement in oxygen lattices

NASA Astrophysics Data System (ADS)

Kamioka, K.; Oga, T.; Izawa, Y.; Kuriyama, K.; Kushida, K.; Kinomura, A.

2014-08-01

The displacement of oxygen lattices in Ge ion-implanted ZnO bulk single crystals is studied by nuclear reaction analysis (NAR), photoluminescence (PL), and Van der Pauw methods. The Ge ion-implantation (net concentration: 2.6 × 1020 cm-3) into ZnO is performed using a multiple-step energy. The high resistivity of ∼103 Ω cm in un-implanted samples remarkably decreased to ∼10-2 Ω cm after implanting Ge-ion and annealing subsequently. NRA measurements of as-implanted and annealed samples suggest the existence of the lattice displacement of O atoms acting as acceptor defects. As O related defects still remain after annealing, these defects are not attributed to the origin of the low resistivity in 800 and 1000 °C annealed ZnO.

Solid-phase microextraction fiber development for sampling and analysis of volatile organohalogen compounds in air.

PubMed

Attari, Seyed Ghavameddin; Bahrami, Abdolrahman; Shahna, Farshid Ghorbani; Heidari, Mahmoud

2014-01-01

A green, environmental friendly and sensitive method for determination of volatile organohalogen compounds was described in this paper. The method is based on a homemade sol-gel single-walled carbon nanotube/silica composite coated solid-phase microextraction to develop for sampling and analysis of Carbon tetrachloride, Benzotrichloride, Chloromethyl methyl ether and Trichloroethylene in air. Application of this method was investigated under different laboratory conditions. Predetermined concentrations of each analytes were prepared in a home-made standard chamber and the influences of experimental parameters such as temperature, humidity, extraction time, storage time, desorption temperature, desorption time and the sorbent performance were investigated. Under optimal conditions, the use of single-walled carbon nanotube/silica composite fiber showed good performance, high sensitive and fast sampling of volatile organohalogen compounds from air. For linearity test the regression correlation coefficient was more than 98% for analyte of interest and linear dynamic range for the proposed fiber and the applied Gas Chromatography-Flame Ionization Detector technique was from 1 to 100 ngmL(-1). Method detection limits ranged between 0.09 to 0.2 ngmL(-1) and method quantification limits were between 0.25 and 0.7 ngmL(-1). Single-walled carbon nanotube/silica composite fiber was highly reproducible, relative standard deviations were between 4.3 to 11.7 percent.

Warfarin genotyping in a single PCR reaction for microchip electrophoresis.

PubMed

Poe, Brian L; Haverstick, Doris M; Landers, James P

2012-04-01

Warfarin is the most commonly prescribed oral anticoagulant medication but also is the second leading cause of emergency room visits for adverse drug reactions. Genetic testing for warfarin sensitivity may reduce hospitalization rates, but prospective genotyping is impeded in part by the turnaround time and costs of genotyping. Microfluidics-based assays can reduce reagent consumption and analysis time; however, no current assay has integrated multiplexed allele-specific PCR for warfarin genotyping with electrophoretic microfluidics hardware. Ideally, such an assay would use a single PCR reaction and, without further processing, a single microchip electrophoresis (ME) run to determine the 3 single-nucleotide polymorphisms (SNPs) affecting warfarin sensitivity [i.e., CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) *2, CYP2C9 *3, and the VKORC1 (vitamin K epoxide reductase complex 1) A/B haplotype]. We designed and optimized primers for a fully multiplexed assay to examine 3 biallelic SNPs with the tetraprimer amplification refractory mutation system (T-ARMS). The assay was developed with conventional PCR equipment and demonstrated for microfluidic infrared-mediated PCR. Genotypes were determined by ME on the basis of the pattern of PCR products. Thirty-five samples of human genomic DNA were analyzed with this multiplex T-ARMS assay, and 100% of the genotype determinations agreed with the results obtained by other validated methods. The sample population included several genotypes conferring warfarin sensitivity, with both homozygous and heterozygous genotypes for each SNP. Total analysis times for the PCR and ME were approximately 75 min (1-sample run) and 90 min (12-sample run). This multiplexed T-ARMS assay coupled with microfluidics hardware constitutes a promising avenue for an inexpensive and rapid platform for warfarin genotyping.

Single-cell regulome data analysis by SCRAT.

PubMed

Ji, Zhicheng; Zhou, Weiqiang; Ji, Hongkai

2017-09-15

Emerging single-cell technologies (e.g. single-cell ATAC-seq, DNase-seq or ChIP-seq) have made it possible to assay regulome of individual cells. Single-cell regulome data are highly sparse and discrete. Analyzing such data is challenging. User-friendly software tools are still lacking. We present SCRAT, a Single-Cell Regulome Analysis Toolbox with a graphical user interface, for studying cell heterogeneity using single-cell regulome data. SCRAT can be used to conveniently summarize regulatory activities according to different features (e.g. gene sets, transcription factor binding motif sites, etc.). Using these features, users can identify cell subpopulations in a heterogeneous biological sample, infer cell identities of each subpopulation, and discover distinguishing features such as gene sets and transcription factors that show different activities among subpopulations. SCRAT is freely available at https://zhiji.shinyapps.io/scrat as an online web service and at https://github.com/zji90/SCRAT as an R package. hji@jhu.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Gluten-containing grains skew gluten assessment in oats due to sample grind non-homogeneity.

PubMed

Fritz, Ronald D; Chen, Yumin; Contreras, Veronica

2017-02-01

Oats are easily contaminated with gluten-rich kernels of wheat, rye and barley. These contaminants are like gluten 'pills', shown here to skew gluten analysis results. Using R-Biopharm R5 ELISA, we quantified gluten in gluten-free oatmeal servings from an in-market survey. For samples with a 5-20ppm reading on a first test, replicate analyses provided results ranging <5ppm to >160ppm. This suggests sample grinding may inadequately disperse gluten to allow a single accurate gluten assessment. To ascertain this, and characterize the distribution of 0.25-g gluten test results for kernel contaminated oats, twelve 50g samples of pure oats, each spiked with a wheat kernel, showed that 0.25g test results followed log-normal-like distributions. With this, we estimate probabilities of mis-assessment for a 'single measure/sample' relative to the <20ppm regulatory threshold, and derive an equation relating the probability of mis-assessment to sample average gluten content. Copyright © 2016 Elsevier Ltd. All rights reserved.

Increasing the accuracy and scalability of the Immunofluorescence Assay for Epstein Barr Virus by inferring continuous titers from a single sample dilution.

PubMed

Goh, Sherry Meow Peng; Swaminathan, Muthukaruppan; Lai, Julian U-Ming; Anwar, Azlinda; Chan, Soh Ha; Cheong, Ian

2017-01-01

High Epstein Barr Virus (EBV) titers detected by the indirect Immunofluorescence Assay (IFA) are a reliable predictor of Nasopharyngeal Carcinoma (NPC). Despite being the gold standard for serological detection of NPC, the IFA is limited by scaling bottlenecks. Specifically, 5 serial dilutions of each patient sample must be prepared and visually matched by an evaluator to one of 5 discrete titers. Here, we describe a simple method for inferring continuous EBV titers from IFA images acquired from NPC-positive patient sera using only a single sample dilution. In the first part of our study, 2 blinded evaluators used a set of reference titer standards to perform independent re-evaluations of historical samples with known titers. Besides exhibiting high inter-evaluator agreement, both evaluators were also in high concordance with historical titers, thus validating the accuracy of the reference titer standards. In the second part of the study, the reference titer standards were IFA-processed and assigned an 'EBV Score' using image analysis. A log-linear relationship between titers and EBV Score was observed. This relationship was preserved even when images were acquired and analyzed 3days post-IFA. We conclude that image analysis of IFA-processed samples can be used to infer a continuous EBV titer with just a single dilution of NPC-positive patient sera. This work opens new possibilities for improving the accuracy and scalability of IFA in the context of clinical screening. Copyright © 2016. Published by Elsevier B.V.

Operation of a sampling train for the analysis of environmental species in coal gasification gas-phase process streams

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

Pochan, M.J.; Massey, M.J.

1979-02-01

This report discusses the results of actual raw product gas sampling efforts and includes: Rationale for raw product gas sampling efforts; design and operation of the CMU gas sampling train; development and analysis of a sampling train data base; and conclusions and future application of results. The results of sampling activities at the CO/sub 2/-Acceptor and Hygas pilot plants proved that: The CMU gas sampling train is a valid instrument for characterization of environmental parameters in coal gasification gas-phase process streams; depending on the particular process configuration, the CMU gas sampling train can reduce gasifier effluent characterization activity to amore » single location in the raw product gas line; and in contrast to the slower operation of the EPA SASS Train, CMU's gas sampling train can collect representative effluent data at a rapid rate (approx. 2 points per hour) consistent with the rate of change of process variables, and thus function as a tool for process engineering-oriented analysis of environmental characteristics.« less

HS-SPME-GC-MS/MS Method for the Rapid and Sensitive Quantitation of 2-Acetyl-1-pyrroline in Single Rice Kernels.

PubMed

Hopfer, Helene; Jodari, Farman; Negre-Zakharov, Florence; Wylie, Phillip L; Ebeler, Susan E

2016-05-25

Demand for aromatic rice varieties (e.g., Basmati) is increasing in the US. Aromatic varieties typically have elevated levels of the aroma compound 2-acetyl-1-pyrroline (2AP). Due to its very low aroma threshold, analysis of 2AP provides a useful screening tool for rice breeders. Methods for 2AP analysis in rice should quantitate 2AP at or below sensory threshold level, avoid artifactual 2AP generation, and be able to analyze single rice kernels in cases where only small sample quantities are available (e.g., breeding trials). We combined headspace solid phase microextraction with gas chromatography tandem mass spectrometry (HS-SPME-GC-MS/MS) for analysis of 2AP, using an extraction temperature of 40 °C and a stable isotopologue as internal standard. 2AP calibrations were linear between the concentrations of 53 and 5380 pg/g, with detection limits below the sensory threshold of 2AP. Forty-eight aromatic and nonaromatic, milled rice samples from three harvest years were screened with the method for their 2AP content, and overall reproducibility, observed for all samples, ranged from 5% for experimental aromatic lines to 33% for nonaromatic lines.

A double sealing technique for increasing the precision of headspace-gas chromatographic analysis.

PubMed

Xie, Wei-Qi; Yu, Kong-Xian; Gong, Yi-Xian

2018-01-19

This paper investigates a new double sealing technique for increasing the precision of the headspace gas chromatographic method. The air leakage problem caused by the high pressure in the headspace vial during the headspace sampling process has a great impact to the measurement precision in the conventional headspace analysis (i.e., single sealing technique). The results (using ethanol solution as the model sample) show that the present technique is effective to minimize such a problem. The double sealing technique has an excellent measurement precision (RSD < 0.15%) and accuracy (recovery = 99.1%-100.6%) for the ethanol quantification. The detection precision of the present method was 10-20 times higher than that in earlier HS-GC work that use conventional single sealing technique. The present double sealing technique may open up a new avenue, and also serve as a general strategy for improving the performance (i.e., accuracy and precision) of headspace analysis of various volatile compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

No Metallicity Correlation Associated with the Kepler Dichotomy

NASA Astrophysics Data System (ADS)

Munoz Romero, Carlos Eduardo; Kempton, Eliza

2018-01-01

NASA’s Kepler mission has discovered thousands of planetary systems, ∼ 20% of which are found to host multiple transiting planets. This relative paucity (compared to the high fraction of single transiting systems) is postulated to result from a distinction in the architecture between multi-transiting systems and those hosting a single transiting planet: a phenomenon usually referred to as the Kepler dichotomy. We investigate the hypothesis that external giant planets are the main cause behind the over-abundance of single- relative to multi-transiting systems, which would be signaled by higher metallicities in the former sample. To this end, we perform a statistical analysis on the stellar metallicity distribution with respect to planet multiplicity in the Kepler data. We perform our analysis on a variety of samples taken from a population of 1062 Kepler main sequence planetary hosts, using precisely determined metallicities from the California-Kepler survey. Contrary to some predictions, we do not find a significant difference between the stellar metallicities of the single- and multiple-transiting planet systems. However, we do find a 43% upper bound for systems with a single non-giant planet that could also host a hidden giant planet, based on metallicity considerations. While the presence of external giant planets might be one factor behind the Kepler dichotomy, our results also favor alternative explanations. We suggest that additional radial velocity and direct imaging measurements are necessary to constrain the presence of gas giants in systems with a single transiting planet.

X-Ray analysis of riverbank sediment of the Tisza (Hungary): identification of particles from a mine pollution event

NASA Astrophysics Data System (ADS)

Osán, J.; Kurunczi, S.; Török, S.; Van Grieken, R.

2002-03-01

A serious heavy metal pollution of the Tisza River occurred on March 10, 2000, arising from a mine-dumping site in Romania. Sediment samples were taken from the main riverbed at six sites in Hungary, on March 16, 2000. The objective of this work was to distinguish the anthropogenic and crustal erosion particles in the river sediment. The samples were investigated using both bulk X-ray fluorescence (XRF) and thin-window electron probe microanalysis (EPMA). For EPMA, a reverse Monte Carlo method calculated the quantitative elemental composition of each single sediment particle. A high abundance of pyrite type particles was observed in some of the samples, indicating the influence of the mine dumps. Backscattered electron images proved that the size of particles with a high atomic number matrix was in the range of 2 μm. In other words the pyrites and the heavy elements form either small particles or are fragments of larger agglomerates. The latter are formed during the flotation process of the mines or get trapped to the natural crustal erosion particles. The XRF analysis of pyrite-rich samples always showed much higher Cu, Zn and Pb concentrations than the rest of the samples, supporting the conclusions of the single-particle EPMA results. In the polluted samples, the concentration of Cu, Zn and Pb reached 0.1, 0.3 and 0.2 wt.%, respectively. As a new approach, the abundance of particle classes obtained from single-particle EPMA and the elemental concentration obtained by XRF were merged into one data set. The dimension of the common data set was reduced by principal component analysis. The first component was determined by the abundance of pyrite and zinc sulfide particles and the concentration of Cu, Zn and Pb. The polluted samples formed a distinct group in the principal component space. The same result was supported by powder diffraction data. These analytical data combined with Earth Observation Techniques can be further used to estimate the quantity of particles originating from mine tailings on a defined river section.

Review of methods to probe single cell metabolism and bioenergetics

DOE PAGES

Vasdekis, Andreas E.; Stephanopoulos, Gregory

2014-10-31

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

Single and two-shot quantitative phase imaging using Hilbert-Huang Transform based fringe pattern analysis

NASA Astrophysics Data System (ADS)

Trusiak, Maciej; Micó, Vicente; Patorski, Krzysztof; García-Monreal, Javier; Sluzewski, Lukasz; Ferreira, Carlos

2016-08-01

In this contribution we propose two Hilbert-Huang Transform based algorithms for fast and accurate single-shot and two-shot quantitative phase imaging applicable in both on-axis and off-axis configurations. In the first scheme a single fringe pattern containing information about biological phase-sample under study is adaptively pre-filtered using empirical mode decomposition based approach. Further it is phase demodulated by the Hilbert Spiral Transform aided by the Principal Component Analysis for the local fringe orientation estimation. Orientation calculation enables closed fringes efficient analysis and can be avoided using arbitrary phase-shifted two-shot Gram-Schmidt Orthonormalization scheme aided by Hilbert-Huang Transform pre-filtering. This two-shot approach is a trade-off between single-frame and temporal phase shifting demodulation. Robustness of the proposed techniques is corroborated using experimental digital holographic microscopy studies of polystyrene micro-beads and red blood cells. Both algorithms compare favorably with the temporal phase shifting scheme which is used as a reference method.

Electro-focusing liquid extractive surface analysis (EF-LESA) coupled to mass spectrometry.

PubMed

Brenton, A Gareth; Godfrey, A Ruth

2014-04-01

Analysis of the chemical composition of surfaces by liquid sampling devices interfaced to mass spectrometry is attractive as the sample stream can be continuously monitored at good sensitivity and selectivity. A sampling probe has been constructed that takes discrete liquid samples (typically <100 nL) of a surface. It incorporates an electrostatic lens system, comprising three electrodes, to which static and pulsed voltages are applied to form a conical "liquid tip", employed to dissolve analytes at a surface. A prototype system demonstrates spatial resolution of 0.093 mm(2). Time of contact between the liquid tip and the surface is controlled to standardize extraction. Calibration graphs of different analyte concentrations on a stainless surface have been measured, together with the probe's reproducibility, carryover, and recovery. A leucine enkephalin-coated surface demonstrated good linearity (R(2) = 0.9936), with a recovery of 90% and a limit of detection of 38 fmol per single spot sampled. The probe is compact and can be fitted into automated sample analysis equipment having potential for rapid analysis of surfaces at a good spatial resolution.

Electro-Focusing Liquid Extractive Surface Analysis (EF-LESA) Coupled to Mass Spectrometry

PubMed Central

2014-01-01

Analysis of the chemical composition of surfaces by liquid sampling devices interfaced to mass spectrometry is attractive as the sample stream can be continuously monitored at good sensitivity and selectivity. A sampling probe has been constructed that takes discrete liquid samples (typically <100 nL) of a surface. It incorporates an electrostatic lens system, comprising three electrodes, to which static and pulsed voltages are applied to form a conical “liquid tip”, employed to dissolve analytes at a surface. A prototype system demonstrates spatial resolution of 0.093 mm2. Time of contact between the liquid tip and the surface is controlled to standardize extraction. Calibration graphs of different analyte concentrations on a stainless surface have been measured, together with the probe’s reproducibility, carryover, and recovery. A leucine enkephalin-coated surface demonstrated good linearity (R2 = 0.9936), with a recovery of 90% and a limit of detection of 38 fmol per single spot sampled. The probe is compact and can be fitted into automated sample analysis equipment having potential for rapid analysis of surfaces at a good spatial resolution. PMID:24597530

Students' Appreciation of Expectation and Variation as a Foundation for Statistical Understanding

ERIC Educational Resources Information Center

Watson, Jane M.; Callingham, Rosemary A.; Kelly, Ben A.

2007-01-01

This study presents the results of a partial credit Rasch analysis of in-depth interview data exploring statistical understanding of 73 school students in 6 contextual settings. The use of Rasch analysis allowed the exploration of a single underlying variable across contexts, which included probability sampling, representation of temperature…

Content Analysis of Papers Submitted to "Communications in Information Literacy," 2007-2013

ERIC Educational Resources Information Center

Hollister, Christopher V.

2014-01-01

The author conducted a content analysis of papers submitted to the journal, "Communications in Information Literacy," from the years 2007-2013. The purpose was to investigate and report on the overall quality characteristics of a statistically significant sample of papers submitted to a single-topic, open access, library and information…

Analysis Tools (AT)

Treesearch

Larry J. Gangi

2006-01-01

The FIREMON Analysis Tools program is designed to let the user perform grouped or ungrouped summary calculations of single measurement plot data, or statistical comparisons of grouped or ungrouped plot data taken at different sampling periods. The program allows the user to create reports and graphs, save and print them, or cut and paste them into a word processor....

Optimal Scaling of HIV-Related Sexual Risk Behaviors in Ethnically Diverse Homosexually Active Men.

ERIC Educational Resources Information Center

Cochran, Susan D.; And Others

1995-01-01

Used homogeneity analysis and latent class analysis to analyze sexual behavior patterns in two samples of homosexually active men. Results support the existence of a single, nonlinear, latent dimension underlying male homosexual behaviors consistent with HIV-related risk taking, providing an efficient means to scale sexual behavior patterns. (RJM)

Analysis of glycosaminoglycan-derived disaccharides by capillary electrophoresis using laser-induced fluorescence detection

PubMed Central

Chang, Yuqing; Yang, Bo; Zhao, Xue; Linhardt, Robert J.

2012-01-01

A quantitative and highly sensitive method for the analysis of glycosaminoglycan (GAG)-derived disaccharides is presented that relies on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. This method enables complete separation of seventeen GAG-derived disaccharides in a single run. Unsaturated disaccharides were derivatized with 2-aminoacridone (AMAC) to improve sensitivity. The limit of detection was at the attomole level and about 100-fold more sensitive than traditional CE-ultraviolet detection. A CE separation timetable was developed to achieve complete resolution and shorten analysis time. The RSD of migration time and peak areas at both low and high concentrations of unsaturated disaccharides are all less than 2.7% and 3.2%, respectively, demonstrating that this is a reproducible method. This analysis was successfully applied to cultured Chinese hamster ovary cell samples for determination of GAG disaccharides. The current method simplifies GAG extraction steps, and reduces inaccuracy in calculating ratios of heparin/heparan sulfate to chondroitin sulfate/dermatan sulfate, resulting from the separate analyses of a single sample. PMID:22609076

An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

NASA Astrophysics Data System (ADS)

Wollman, Adam J. M.; Miller, Helen; Foster, Simon; Leake, Mark C.

2016-10-01

Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells.

User's Guide for MSAP2D: A Program for Unsteady Aerodynamic and Aeroelastic (Flutter and Forced Response) Analysis of Multistage Compressors and Turbines. 1.0

NASA Technical Reports Server (NTRS)

Reddy, T. S. R.; Srivastava, R.

1996-01-01

This guide describes the input data required for using MSAP2D (Multi Stage Aeroelastic analysis Program - Two Dimensional) computer code. MSAP2D can be used for steady, unsteady aerodynamic, and aeroelastic (flutter and forced response) analysis of bladed disks arranged in multiple blade rows such as those found in compressors, turbines, counter rotating propellers or propfans. The code can also be run for single blade row. MSAP2D code is an extension of the original NPHASE code for multiblade row aerodynamic and aeroelastic analysis. Euler equations are used to obtain aerodynamic forces. The structural dynamic equations are written for a rigid typical section undergoing pitching (torsion) and plunging (bending) motion. The aeroelastic equations are solved in time domain. For single blade row analysis, frequency domain analysis is also provided to obtain unsteady aerodynamic coefficients required in an eigen analysis for flutter. In this manual, sample input and output are provided for a single blade row example, two blade row example with equal and unequal number of blades in the blade rows.

A Facile Droplet-Chip-Time-Resolved Inductively Coupled Plasma Mass Spectrometry Online System for Determination of Zinc in Single Cell.

PubMed

Wang, Han; Chen, Beibei; He, Man; Hu, Bin

2017-05-02

Single cell analysis is a significant research field in recent years reflecting the heterogeneity of cells in a biological system. In this work, a facile droplet chip was fabricated and online combined with time-resolved inductively coupled plasma mass spectrometry (ICPMS) via a microflow nebulizer for the determination of zinc in single HepG2 cells. On the focusing geometric designed PDMS microfluidic chip, the aqueous cell suspension was ejected and divided by hexanol to generate droplets. The droplets encapsulated single cells remain intact during the transportation into ICP for subsequent detection. Under the optimized conditions, the frequency of droplet generation is 3-6 × 10 6 min -1 , and the injected cell number is 2500 min -1 , which can ensure the single cell encapsulation. ZnO nanoparticles (NPs) were used for the quantification of zinc in single cells, and the accuracy was validated by conventional acid digestion-ICPMS method. The ZnO NPs incubated HepG2 cells were analyzed as model samples, and the results exhibit the heterogeneity of HepG2 cells in the uptake/adsorption of ZnO NPs. The developed online droplet-chip-ICPMS analysis system achieves stable single cell encapsulation and has high throughput for single cell analysis. It has the potential in monitoring the content as well as distribution of trace elements/NPs at the single cell level.

Nine-analyte detection using an array-based biosensor

NASA Technical Reports Server (NTRS)

Taitt, Chris Rowe; Anderson, George P.; Lingerfelt, Brian M.; Feldstein, s. Mark. J.; Ligler, Frances S.

2002-01-01

A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 x 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersiniapestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected.

Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity

NASA Astrophysics Data System (ADS)

Wahl, Michael; Rahn, Hans-Juergen; Ortmann, Uwe; Erdmann, Rainer; Boehmer, Martin; Enderlein, Joerg

2002-03-01

Fluorescence lifetime measurement of organic fluorophores is a powerful tool for distinguishing molecules of interest from background or other species. This is of interest in sensitive analysis and Single Molecule Detection (SMD). A demand in many applications is to provide 2-D imaging together with lifetime information. The method of choice is then Time-Correlated Single Photon Counting (TCSPC). We have devloped a compact system on a single PC board that can perform TCSPC at high throughput, while synchronously driving a piezo scanner holding the immobilized sample. The system allows count rates up to 3 MHz and a resolution down to 30 ps. An overall Instrument Response Function down to 300ps is achieved with inexpensive detectors and diode lasers. The board is designed for the PCI bus, permitting high throughput without loss of counts. It is reconfigurable to operate in different modes. The Time-Tagged Time-Resolved (TTTR) mode permits the recording of all photon events with a real-time tag allowing data analysis with unlimited flexibility. We use the Time-Tag clock for an external piezo scanner that moves the sample. As the clock source is common for scanning and tagging, the individual photons can be matched to pixels. Demonstrating the capablities of the system we studied single molecule solutions. Lifetime imaging can be performed at high resolution with as few as 100 photons per pixel.

Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils

DOE PAGES

Eichorst, Stephanie A.; Strasser, Florian; Woyke, Tanja; ...

2015-08-31

The combined approach of incubating environmental samples with stable isotope-labeled substrates followed by single-cell analyses through high-resolution secondary ion mass spectrometry (NanoSIMS) or Raman microspectroscopy provides insights into the in situ function of microorganisms. This approach has found limited application in soils presumably due to the dispersal of microbial cells in a large background of particles. We developed a pipeline for the efficient preparation of cell extracts from soils for subsequent single-cell methods by combining cell detachment with separation of cells and soil particles followed by cell concentration. The procedure was evaluated by examining its influence on cell recoveries andmore » microbial community composition across two soils. This approach generated a cell fraction with considerably reduced soil particle load and of sufficient small size to allow single-cell analysis by NanoSIMS, as shown when detecting active N2-fixing and cellulose-responsive microorganisms via 15N2 and 13C-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a 13C-labeled carbon source and deuterium oxide (D2O, a general activity marker). Lastly, the described sample preparation procedure enables single-cell analysis of soil microorganisms using NanoSIMS and Raman microspectroscopy, but should also facilitate single-cell sorting and sequencing.« less

Chemical analyses of provided samples

NASA Technical Reports Server (NTRS)

Becker, Christopher H.

1993-01-01

A batch of four samples were received and chemical analysis was performed of the surface and near surface regions of the samples by the surface analysis by laser ionization (SALI) method. The samples included four one-inch diameter optics labeled windows no. PR14 and PR17 and MgF2 mirrors 9-93 PPPC exp. and control DMES 26-92. The analyses emphasized surface contamination or modification. In these studies, pulsed desorption by 355 nm laser light and single-photon ionization (SPI) above the sample by coherent 118 nm radiation (at approximately 5 x 10(exp 5) W/cm(sup 2)) were used, emphasizing organic analysis. For the two windows with an apparent yellowish contaminant film, higher desorption laser power was needed to provide substantial signals, indicating a less volatile contamination than for the two mirrors. Window PR14 and the 9-93 mirror showed more hydrocarbon components than the other two samples. The mass spectra, which show considerable complexity, are discussed in terms of various potential chemical assignments.

Nuts and Bolts - Techniques for Genesis Sample Curation

NASA Technical Reports Server (NTRS)

Burkett, Patti J.; Rodriquez, M. C.; Allton, J. H.

2011-01-01

The Genesis curation staff at NASA Johnson Space Center provides samples and data for analysis to the scientific community, following allocation approval by the Genesis Oversight Committee, a sub-committee of CAPTEM (Curation Analysis Planning Team for Extraterrestrial Materials). We are often asked by investigators within the scientific community how we choose samples to best fit the requirements of the request. Here we will demonstrate our techniques for characterizing samples and satisfying allocation requests. Even with a systematic approach, every allocation is unique. We are also providing updated status of the cataloging and characterization of solar wind collectors as of January 2011. The collection consists of 3721 inventoried samples consisting of a single fragment, or multiple fragments containerized or pressed between post-it notes, jars or vials of various sizes.

A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications.

PubMed

Haque, Ashraful; Engel, Jessica; Teichmann, Sarah A; Lönnberg, Tapio

2017-08-18

RNA sequencing (RNA-seq) is a genomic approach for the detection and quantitative analysis of messenger RNA molecules in a biological sample and is useful for studying cellular responses. RNA-seq has fueled much discovery and innovation in medicine over recent years. For practical reasons, the technique is usually conducted on samples comprising thousands to millions of cells. However, this has hindered direct assessment of the fundamental unit of biology-the cell. Since the first single-cell RNA-sequencing (scRNA-seq) study was published in 2009, many more have been conducted, mostly by specialist laboratories with unique skills in wet-lab single-cell genomics, bioinformatics, and computation. However, with the increasing commercial availability of scRNA-seq platforms, and the rapid ongoing maturation of bioinformatics approaches, a point has been reached where any biomedical researcher or clinician can use scRNA-seq to make exciting discoveries. In this review, we present a practical guide to help researchers design their first scRNA-seq studies, including introductory information on experimental hardware, protocol choice, quality control, data analysis and biological interpretation.

Determination of metals in lubricating oils by flame atomic absorption spectrometry using a single-bore high-pressure pneumatic nebulizer.

PubMed

Mora, J; Todolí, J L; Sempere, F J; Canals, A; Hernandis, V

2000-12-01

The behaviour of a single-bore high-pressure pneumatic nebulizer (SBHPPN) as a tool for the analysis of lubricating oils by flame atomic absorption spectrometry (FAAS) was investigated. The effects of the sample oil content [from 10% to 100% (w/w) oil in 4-methylpentan-2-one, IBMK] and the carrier nature (IBMK and methanol) on the characteristics of the aerosols generated, on the analyte transport efficiency and on the analytical figures of merit in FAAS were studied. A pneumatic concentric nebulizer (PCN) was used for comparison. Increasing the oil content increases the viscosity of the sample. With the PCN this gives rise to coarser aerosols, making it impossible to nebulize samples with an oil content higher than 70% (w/w). Using the SBHPPN, the viscosity of the sample scarcely affects the characteristics of the primary aerosols. Hence, the SBHPPN is able, by using the appropriate carrier, to nebulize pure lubricating oils. Among the carriers tested, IBMK is the most advisable because it is fully miscible with all the oil samples. The SBHPPN provides higher sensitivities and lower limits of detection than the PCN. Compared with a method based on organic dilution, the use of the SBHPPN for the direct analysis of lubricating oils by FAAS makes it possible, in addition to increasing the analysis throughput, to detect elements at lower concentrations. Moreover, the SBHPPN provides similar results to those obtained using a previous acid digestion step.

Direct analysis of environmental and biological samples for total mercury with comparison of sequential atomic absorption and fluorescence measurements from a single combustion event

NASA Astrophysics Data System (ADS)

Cizdziel, James V.; Tolbert, Candice; Brown, Garry

2010-02-01

A Direct Mercury Analyzer (DMA) based on sample combustion, concentration of mercury by amalgamation with gold, and cold vapor atomic absorption spectrometry (CVAAS) was coupled to a mercury-specific cold vapor atomic fluorescence spectrometer (CVAFS). The purpose was to evaluate combustion-AFS, a technique which is not commercially available, for low-level analysis of mercury in environmental and biological samples. The experimental setup allowed for comparison of dual measurements of mercury (AAS followed by AFS) for a single combustion event. The AFS instrument control program was modified to properly time capture of mercury from the DMA, avoiding deleterious combustion products from reaching its gold traps. Calibration was carried out using both aqueous solutions and solid reference materials. The absolute detection limits for mercury were 0.002 ng for AFS and 0.016 ng for AAS. Recoveries for reference materials ranged from 89% to 111%, and the precision was generally found to be <10% relative standard deviation (RSD). The two methods produced similar results for samples of hair, finger nails, coal, soil, leaves and food stuffs. However, for samples with mercury near the AAS detection limit (e.g., filter paper spotted with whole blood and segments of tree rings) the signal was still quantifiable with AFS, demonstrating the lower detection limit and greater sensitivity of AFS. This study shows that combustion-AFS is feasible for the direct analysis of low levels of mercury in solid samples that would otherwise require time-consuming and contamination-prone digestion.

Quality Assessment of Kumu Injection, a Traditional Chinese Medicine Preparation, Using HPLC Combined with Chemometric Methods and Qualitative and Quantitative Analysis of Multiple Alkaloids by Single Marker.

PubMed

Wang, Ning; Li, Zhi-Yong; Zheng, Xiao-Li; Li, Qiao; Yang, Xin; Xu, Hui

2018-04-09

Kumu injection (KMI) is a common-used traditional Chinese medicine (TCM) preparation made from Picrasma quassioides (D. Don) Benn. rich in alkaloids. An innovative technique for quality assessment of KMI was developed using high performance liquid chromatography (HPLC) combined with chemometric methods and qualitative and quantitative analysis of multi-components by single marker (QAMS). Nigakinone (PQ-6, 5-hydroxy-4-methoxycanthin-6-one), one of the most abundant alkaloids responsible for the major pharmacological activities of Kumu, was used as a reference substance. Six alkaloids in KMI were quantified, including 6-hydroxy- β -carboline-1-carboxylic acid (PQ-1), 4,5-dimethoxycanthin-6-one (PQ-2), β -carboline-1-carboxylic acid (PQ-3), β -carboline-1-propanoic acid (PQ-4), 3-methylcanthin-5,6-dione (PQ-5), and PQ-6. Based on the outcomes of twenty batches of KMI samples, the contents of six alkaloids were used for further chemometric analysis. By hierarchical cluster analysis (HCA), radar plots, and principal component analysis (PCA), all the KMI samples could be categorized into three groups, which were closely related to production date and indicated the crucial influence of herbal raw material on end products of KMI. QAMS combined with chemometric analysis could accurately measure and clearly distinguish the different quality samples of KMI. Hence, QAMS is a feasible and promising method for the quality control of KMI.

Crystal structure, spectral, thermal and dielectric studies of a new zinc benzoate single crystal

NASA Astrophysics Data System (ADS)

Bijini, B. R.; Prasanna, S.; Deepa, M.; Nair, C. M. K.; Rajendra Babu, K.

2012-11-01

Single crystals of zinc benzoate with a novel structure were grown in gel media. Sodium metasilicate of gel density 1.04 g/cc at pH 6 was employed to yield transparent single crystals. The crystal structure of the compound was ascertained by single crystal X-ray diffractometry. It was noted that the crystal belongs to monoclinic system with space group P21/c with unit cell parameters a = 10.669(1) Å, b = 12.995(5) Å, c = 19.119(3) Å, and β = 94.926(3)°. The crystal was seen to possess a linear polymeric structure along b-axis; with no presence of coordinated or lattice water. CHN analysis established the stoichiometric composition of the crystal. The existence of functional groups present in the single crystal system was confirmed by FT-IR studies. The thermal characteristic of the sample was analysed by TGA-DTA techniques, and the sample was found to be thermally stable up to 280 °C. The kinetic and thermodynamic parameters were also determined. UV-Vis spectroscopy corroborated the transparency of the crystal and revealed the optical band gap to be 4 eV. Dielectric studies showed decrease in the dielectric constant of the sample with increase in frequency.

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

PubMed Central

Turm, Hagit; Mukherjee, Diptendu; Haritan, Doron; Tahor, Maayan; Citri, Ami

2014-01-01

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2. In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells. The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies. PMID:25225819

Analysis of Phase Separation in Czochralski Grown Single Crystal Ilmenite

NASA Technical Reports Server (NTRS)

Wilkins, R.; Powell, Kirk St. A.; Loregnard, Kieron R.; Lin, Sy-Chyi; Muthusami, Jayakumar; Zhou, Feng; Pandey, R. K.; Brown, Geoff; Hawley, M. E.

1998-01-01

Ilmenite (FeTiOs) is a wide bandgap semiconductor with an energy gap of 2.58 eV. Ilmenite has properties suited for radiation tolerant applications, as well as a variety of other electronic applications. Single crystal ilmenite has been grown from the melt using the Czochralski method. Growth conditions have a profound effect on the microstructure of the samples. Here we present data from a variety of analytical techniques which indicate that some grown crystals exhibit distinct phase separation during growth. This phase separation is apparent for both post-growth annealed and unannealed samples. Under optical microscopy, there appear two distinct areas forming a matrix with an array of dots on order of 5 pm diameter. While appearing bright in the optical micrograph, atomic force microscope (AFM) shows the dots to be shallow pits on the surface. Magnetic force microscope (MFM) shows the dots to be magnetic. Phase identification via electron microprobe analysis (EMPA) indicates two major phases in the unannealed samples and four in the annealed samples, where the dots appear to be almost pure iron. This is consistent with micrographs taken with a scanning probe microscope used in the magnetic force mode. Samples that do not exhibit the phase separation have little or no discernible magnetic structure detectable by the MFM.

The Beck Depression Inventory, Second Edition (BDI-II): A Cross-Sample Structural Analysis

ERIC Educational Resources Information Center

Strunk, Kamden K.; Lane, Forrest C.

2017-01-01

A common concern about the Beck Depression Inventory, Second edition (BDI-II) among researchers in the area of depression has long been the single-factor scoring scheme. Methods exist for making cross-sample comparisons of latent structure but tend to rely on estimation methods that can be imprecise and unnecessarily complex. This study presents a…

Application of higher harmonic blade feathering for helicopter vibration reduction

NASA Technical Reports Server (NTRS)

Powers, R. W.

1978-01-01

Higher harmonic blade feathering for helicopter vibration reduction is considered. Recent wind tunnel tests confirmed the effectiveness of higher harmonic control in reducing articulated rotor vibratory hub loads. Several predictive analyses developed in support of the NASA program were shown to be capable of calculating single harmonic control inputs required to minimize a single 4P hub response. In addition, a multiple-input, multiple-output harmonic control predictive analysis was developed. All techniques developed thus far obtain a solution by extracting empirical transfer functions from sampled data. Algorithm data sampling and processing requirements are minimal to encourage adaptive control system application of such techniques in a flight environment.

A transmission imaging spectrograph and microfabricated channel system for DNA analysis.

PubMed

Simpson, J W; Ruiz-Martinez, M C; Mulhern, G T; Berka, J; Latimer, D R; Ball, J A; Rothberg, J M; Went, G T

2000-01-01

In this paper we present the development of a DNA analysis system using a microfabricated channel device and a novel transmission imaging spectrograph which can be efficiently incorporated into a high throughput genomics facility for both sizing and sequencing of DNA fragments. The device contains 48 channels etched on a glass substrate. The channels are sealed with a flat glass plate which also provides a series of apertures for sample loading and contact with buffer reservoirs. Samples can be easily loaded in volumes up to 640 nL without band broadening because of an efficient electrokinetic stacking at the electrophoresis channel entrance. The system uses a dual laser excitation source and a highly sensitive charge-coupled device (CCD) detector allowing for simultaneous detection of many fluorescent dyes. The sieving matrices for the separation of single-stranded DNA fragments are polymerized in situ in denaturing buffer systems. Examples of separation of single-stranded DNA fragments up to 500 bases in length are shown, including accurate sizing of GeneCalling fragments, and sequencing samples prepared with a reduced amount of dye terminators. An increase in sample throughput has been achieved by color multiplexing.

Chemical analyses of provided samples

NASA Technical Reports Server (NTRS)

Becker, Christopher H.

1993-01-01

Two batches of samples were received and chemical analysis was performed of the surface and near surface regions of the samples by the surface analysis by laser ionization (SALI) method. The samples included four one-inch optics and several paint samples. The analyses emphasized surface contamination or modification. In these studies, pulsed sputtering by 7 keV Ar+ and primarily single-photon ionization (SPI) by coherent 118 nm radiation (at approximately 5 x 10(exp 5) W/cm(sup 2) were used. For two of the samples, also multiphoton ionization (MPI) at 266 nm (approximately 5 x 10(exp 11) W/cm(sup 2) was used. Most notable among the results was the silicone contamination on Mg2 mirror 28-92, and that the Long Duration Exposure Facility (LDEF) paint sample had been enriched in K and Na and depleted in Zn, Si, B, and organic compounds relative to the control paint.

Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing.

PubMed

Salvianti, Francesca; Rotunno, Giada; Galardi, Francesca; De Luca, Francesca; Pestrin, Marta; Vannucchi, Alessandro Maria; Di Leo, Angelo; Pazzagli, Mario; Pinzani, Pamela

2015-09-01

The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch(®) and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer. Upon whole genome amplification, samples were analysed by NGS on the Ion Torrent PGM™ system (Life Technologies) using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies), designed to investigate genomic "hot spot" regions of 50 oncogenes and tumor suppressor genes. We successfully sequenced five single cells, a pool of 5 cells and DNA from a cellular pellet of the same cell line with a mean depth of the sequencing reaction ranging from 1581 to 3479 reads. We found 27 sequence variants in 18 genes, 15 of which already reported in the COSMIC or dbSNP databases. We confirmed the presence of two somatic mutations, in the BRAF and TP53 gene, which had been already reported for this cells line, but also found new mutations and single nucleotide polymorphisms. Three variants were common to all the analysed samples, while 18 were present only in a single cell suggesting a high heterogeneity within the same cell line. This paper presents an optimized workflow for the molecular characterization of multiple genes in single cells by NGS. The described pipeline can be easily transferred to the study of single CTCs from oncologic patients.

An enhanced droplet-based liquid microjunction surface sampling system coupled with HPLC-ESI-MS/MS for spatially resolved analysis

DOE PAGES

Van Berkel, Gary J.; Weiskittel, Taylor M.; Kertesz, Vilmos

2014-11-07

Droplet-based liquid microjunction surface sampling coupled with high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) for spatially resolved analysis provides the possibility of effective analysis of complex matrix samples and can provide a greater degree of chemical information from a single spot sample than is typically possible with a direct analysis of an extract. Described here is the setup and enhanced capabilities of a discrete droplet liquid microjunction surface sampling system employing a commercially available CTC PAL autosampler. The system enhancements include incorporation of a laser distance sensor enabling unattended analysis of samples and sample locations of dramatically disparatemore » height as well as reliably dispensing just 0.5 μL of extraction solvent to make the liquid junction to the surface, wherein the extraction spot size was confined to an area about 0.7 mm in diameter; software modifications improving the spatial resolution of sampling spot selection from 1.0 to 0.1 mm; use of an open bed tray system to accommodate samples as large as whole-body rat thin tissue sections; and custom sample/solvent holders that shorten sampling time to approximately 1 min per sample. Lastly, the merit of these new features was demonstrated by spatially resolved sampling, HPLC separation, and mass spectral detection of pharmaceuticals and metabolites from whole-body rat thin tissue sections and razor blade (“crude”) cut mouse tissue.« less

An enhanced droplet-based liquid microjunction surface sampling system coupled with HPLC-ESI-MS/MS for spatially resolved analysis

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

Van Berkel, Gary J.; Weiskittel, Taylor M.; Kertesz, Vilmos

Droplet-based liquid microjunction surface sampling coupled with high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) for spatially resolved analysis provides the possibility of effective analysis of complex matrix samples and can provide a greater degree of chemical information from a single spot sample than is typically possible with a direct analysis of an extract. Described here is the setup and enhanced capabilities of a discrete droplet liquid microjunction surface sampling system employing a commercially available CTC PAL autosampler. The system enhancements include incorporation of a laser distance sensor enabling unattended analysis of samples and sample locations of dramatically disparatemore » height as well as reliably dispensing just 0.5 μL of extraction solvent to make the liquid junction to the surface, wherein the extraction spot size was confined to an area about 0.7 mm in diameter; software modifications improving the spatial resolution of sampling spot selection from 1.0 to 0.1 mm; use of an open bed tray system to accommodate samples as large as whole-body rat thin tissue sections; and custom sample/solvent holders that shorten sampling time to approximately 1 min per sample. Lastly, the merit of these new features was demonstrated by spatially resolved sampling, HPLC separation, and mass spectral detection of pharmaceuticals and metabolites from whole-body rat thin tissue sections and razor blade (“crude”) cut mouse tissue.« less

Simulations of single-particle imaging of hydrated proteins with x-ray free-electron lasers

NASA Astrophysics Data System (ADS)

Fortmann-Grote, C.; Bielecki, J.; Jurek, Z.; Santra, R.; Ziaja-Motyka, B.; Mancuso, A. P.

2017-08-01

We employ start-to-end simulations to model coherent diffractive imaging of single biomolecules using x-ray free electron lasers. This technique is expected to yield new structural information about biologically relevant macromolecules thanks to the ability to study the isolated sample in its natural environment as opposed to crystallized or cryogenic samples. The effect of the solvent on the diffraction pattern and interpretability of the data is an open question. We present first results of calculations where the solvent is taken into account explicitly. They were performed with a molecular dynamics scheme for a sample consisting of a protein and a hydration layer of varying thickness. Through R-factor analysis of the simulated diffraction patterns from hydrated samples, we show that the scattering background from realistic hydration layers of up to 3 Å thickness presents no obstacle for the resolution of molecular structures at the sub-nm level.

A Generalized Approach to Forensic Dye Identification: Development and Utility of Reference Libraries.

PubMed

Groves, Ethan; Palenik, Skip; Palenik, Christopher S

2018-04-18

While color is arguably the most important optical property of evidential fibers, the actual dyestuffs responsible for its expression in them are, in forensic trace evidence examinations, rarely analyzed and still less often identified. This is due, primarily, to the exceedingly small quantities of dye present in a single fiber as well as to the fact that dye identification is a challenging analytical problem, even when large quantities are available for analysis. Among the practical reasons for this are the wide range of dyestuffs available (and the even larger number of trade names), the low total concentration of dyes in the finished product, the limited amount of sample typically available for analysis in forensic cases, and the complexity of the dye mixtures that may exist within a single fiber. Literature on the topic of dye analysis is often limited to a specific method, subset of dyestuffs, or an approach that is not applicable given the constraints of a forensic analysis. Here, we present a generalized approach to dye identification that ( 1 ) combines several robust analytical methods, ( 2 ) is broadly applicable to a wide range of dye chemistries, application classes, and fiber types, and ( 3 ) can be scaled down to forensic casework-sized samples. The approach is based on the development of a reference collection of 300 commercially relevant textile dyes that have been characterized by a variety of microanalytical methods (HPTLC, Raman microspectroscopy, infrared microspectroscopy, UV-Vis spectroscopy, and visible microspectrophotometry). Although there is no single approach that is applicable to all dyes on every type of fiber, a combination of these analytical methods has been applied using a reproducible approach that permits the use of reference libraries to constrain the identity of and, in many cases, identify the dye (or dyes) present in a textile fiber sample.

Extractive electrospray ionization mass spectrometry toward in situ analysis without sample pretreatment.

PubMed

Li, Ming; Hu, Bin; Li, Jianqiang; Chen, Rong; Zhang, Xie; Chen, Huanwen

2009-09-15

A homemade novel nanoextractive electrospray ionization (nanoEESI) source has been characterized for in situ mass spectrometric analysis of ambient samples without sample pretreatment. The primary ions generated using a nanospray emitter interact with the neutral sample plume created by manually nebulizing liquid samples, allowing production of the analyte ions in the spatial cross section of the nanoEESI source. The performance of nanoEESI is experimentally investigated by coupling the nanoEESI source to a commercial LTQ mass spectrometer for rapid analysis of various ambient samples using positive/negative ion detection modes. Compounds of interest in actual samples such as aerosol drug preparations, beverages, milk suspensions, farmland water, and groundwater were unambiguously detected using tandem nanoEESI ion trap mass spectrometry. The limit of detection was low picogram per milliliter levels for the compounds tested. Acceptable relative standard deviation (RSD) values (5-10%) were obtained for direct measurement of analytes in complex matrixes, providing linear dynamic signal responses using manual sample introduction. A single sample analysis was completed within 1.2 s. Requiring no sheath gas for either primary ion production or neutral sample introduction, the nanoEESI has advantages including readiness for miniaturization and integration, simple maintenance, easy operation, and low cost. The experimental data demonstrate that the nanoEESI is a promising tool for high-throughput, sensitive, quantitative, in situ analysis of ambient complex samples, showing potential applications for in situ analysis in multiple disciplines including but not limited to pharmaceutical analysis, food quality control, pesticides residue detection, and homeland security.

Ar-Ar Thermochronlogy of Apollo 12 Impact-Melt Breccia 12033,638-1

NASA Technical Reports Server (NTRS)

Crow, C. A.; Cassata, W. S.; Jolliff, B. L.; Ziegler, R. A.; Borg, L. E.; Shearer, C. K.

2017-01-01

We have undertaken an Ar-Ar thermochronology investigation as part of a coordinated multichronometer analysis of a single Apollo 12 impact- melt breccia to demonstrate the wide range of information that can be obtained for a single complex rock. This has implications for the age of formation, component makeup, and subsequent impact/shock and exposure history of the sample. This study also serves as a capabilities demonstration for the proposed MoonRise Mission [1]. The goal of this investigation is to elucidate the history of this sample through coordinated 40Ar*/39Ar, Sm-Nd, Rb-Sr and zircon 207Pb-206Pb ages along with geochemical and petrographic context on a relatively small (approximately 450 mg) sample. Here, we report preliminary results of the Ar-Ar thermochronology.

Microchip electrophoresis of oligosaccharides using large-volume sample stacking with an electroosmotic flow pump in a single channel.

PubMed

Kawai, Takayuki; Sueyoshi, Kenji; Kitagawa, Fumihiko; Otsuka, Koji

2010-08-01

The applicability of an online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP), to microchip zone electrophoresis (MCZE) for the analysis of oligosaccharides was investigated. Since the sample stacking and separation proceeded continuously without polarity switching in LVSEP, a single "straight" channel microchip could be employed. In the MCZE analysis of oligosaccharides, sample adsorption onto the channel surface should be suppressed, so the straight microchannel was modified with poly(vinyl alcohol) (PVA). So far, the mechanism of LVSEP in the polymer-coated capillary or microchannel has not been reported, and thus, the LVSEP process in the PVA-coated channel was investigated by fluorescence imaging. Although it is well-known that the PVA coating can suppress the electroosmotic flow (EOF), an enhanced EOF with a mobility of 4.4 x 10(-4) cm(2)/(V x s) was observed in a low ionic strength sample solution. It was revealed that such temporarily enhanced EOF in the sample zone worked as the driving force to remove the sample matrix in LVSEP. To evaluate the analytical performance of LVSEP-MCZE, oligosaccharides were analyzed in the PVA-coated straight channel. As a result, both the glucose ladder and oligosaccharides obtained from bovine ribonuclease B were well enriched and separated with up to 2200-2900-fold sensitivity enhancement compared to those in a conventional MCZE analysis. The run-to-run repeatabilities of the migration time and peak height were good with relative standard deviations of 1.1% and 7.2%, respectively, which were better than those of normal MCZE. By applying the LVSEP technique to MCZE, a complicated voltage program for fluidic control could be simplified from four channels for two steps to two channels for one step.

Genomic Analysis of Complex Microbial Communities in Wounds

DTIC Science & Technology

2012-01-01

thoroughly in the ecology literature. Permutation Multivariate Analysis of Variance ( PerMANOVA ). We used PerMANOVA to test the null-hypothesis of no...difference between the bacterial communities found within a single wound compared to those from different patients (α = 0.05). PerMANOVA is a...permutation-based version of the multivariate analysis of variance (MANOVA). PerMANOVA uses the distances between samples to partition variance and

Nanofluid of zinc oxide nanoparticles in ionic liquid for single drop liquid microextraction of fungicides in environmental waters prior to high performance liquid chromatographic analysis.

PubMed

Amde, Meseret; Tan, Zhi-Qiang; Liu, Rui; Liu, Jing-Fu

2015-05-22

Using a nanofluid obtained by dispersing ZnO nanoparticles (ZnO NPs) in 1-hexyl-3-methylimidazolium hexafluorophosphate, new single drop microextraction method was developed for simultaneous extraction of three fungicides (chlorothalonil, kresoxim-methyl and famoxadone) in water samples prior to their analysis by high performance liquid chromatography (HPLC-VWD). The parameters affecting the extraction efficiency such as amount of ZnO NPs in the nanofluid, solvent volume, extraction time, stirring rate, pH and ionic strength of the sample solution were optimized. Under the optimized conditions, the limits of detection were in the range of 0.13-0.19ng/mL, the precision of the method assessed with intra-day and inter-day relative standard deviations were <4.82% and <7.04%, respectively. The proposed method was successfully applied to determine the three fungicides in real water samples including lake water, river water, as well as effluent and influent of wastewater treatment plant, with recoveries in the range of 74.94-96.11% at 5ng/mL spiking level. Besides to being environmental friendly, the high enrichment factor and the data quality obtained with the proposed method demonstrated its potential for application in multi residue analysis of fungicides in actual water samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Genetic analysis of dyslexia candidate genes in the European cross-linguistic NeuroDys cohort.

PubMed

Becker, Jessica; Czamara, Darina; Scerri, Tom S; Ramus, Franck; Csépe, Valéria; Talcott, Joel B; Stein, John; Morris, Andrew; Ludwig, Kerstin U; Hoffmann, Per; Honbolygó, Ferenc; Tóth, Dénes; Fauchereau, Fabien; Bogliotti, Caroline; Iannuzzi, Stéphanie; Chaix, Yves; Valdois, Sylviane; Billard, Catherine; George, Florence; Soares-Boucaud, Isabelle; Gérard, Christophe-Loïc; van der Mark, Sanne; Schulz, Enrico; Vaessen, Anniek; Maurer, Urs; Lohvansuu, Kaisa; Lyytinen, Heikki; Zucchelli, Marco; Brandeis, Daniel; Blomert, Leo; Leppänen, Paavo H T; Bruder, Jennifer; Monaco, Anthony P; Müller-Myhsok, Bertram; Kere, Juha; Landerl, Karin; Nöthen, Markus M; Schulte-Körne, Gerd; Paracchini, Silvia; Peyrard-Janvid, Myriam; Schumacher, Johannes

2014-05-01

Dyslexia is one of the most common childhood disorders with a prevalence of around 5-10% in school-age children. Although an important genetic component is known to have a role in the aetiology of dyslexia, we are far from understanding the molecular mechanisms leading to the disorder. Several candidate genes have been implicated in dyslexia, including DYX1C1, DCDC2, KIAA0319, and the MRPL19/C2ORF3 locus, each with reports of both positive and no replications. We generated a European cross-linguistic sample of school-age children - the NeuroDys cohort - that includes more than 900 individuals with dyslexia, sampled with homogenous inclusion criteria across eight European countries, and a comparable number of controls. Here, we describe association analysis of the dyslexia candidate genes/locus in the NeuroDys cohort. We performed both case-control and quantitative association analyses of single markers and haplotypes previously reported to be dyslexia-associated. Although we observed association signals in samples from single countries, we did not find any marker or haplotype that was significantly associated with either case-control status or quantitative measurements of word-reading or spelling in the meta-analysis of all eight countries combined. Like in other neurocognitive disorders, our findings underline the need for larger sample sizes to validate possibly weak genetic effects.

Practical limitations of single particle ICP-MS in the determination of nanoparticle size distributions and dissolution: case of rare earth oxides.

PubMed

Fréchette-Viens, Laurie; Hadioui, Madjid; Wilkinson, Kevin J

2017-01-15

The applicability of single particle ICP-MS (SP-ICP-MS) for the analysis of nanoparticle size distributions and the determination of particle numbers was evaluated using the rare earth oxide, La 2 O 3 , as a model particle. The composition of the storage containers, as well as the ICP-MS sample introduction system were found to significantly impact SP-ICP-MS analysis. While La 2 O 3 nanoparticles (La 2 O 3 NP) did not appear to interact strongly with sample containers, adsorptive losses of La 3+ (over 24h) were substantial (>72%) for fluorinated ethylene propylene bottles as opposed to polypropylene (<10%). Furthermore, each part of the sample introduction system (nebulizers made of perfluoroalkoxy alkane (PFA) or glass, PFA capillary tubing, and polyvinyl chloride (PVC) peristaltic pump tubing) contributed to La 3+ adsorptive losses. On the other hand, the presence of natural organic matter in the nanoparticle suspensions led to a decreased adsorptive loss in both the sample containers and the introduction system, suggesting that SP-ICP-MS may nonetheless be appropriate for NP analysis in environmental matrices. Coupling of an ion-exchange resin to the SP-ICP-MS led to more accurate determinations of the La 2 O 3 NP size distributions. Copyright © 2016 Elsevier B.V. All rights reserved.

Damage evolution analysis of coal samples under cyclic loading based on single-link cluster method

NASA Astrophysics Data System (ADS)

Zhang, Zhibo; Wang, Enyuan; Li, Nan; Li, Xuelong; Wang, Xiaoran; Li, Zhonghui

2018-05-01

In this paper, the acoustic emission (AE) response of coal samples under cyclic loading is measured. The results show that there is good positive relation between AE parameters and stress. The AE signal of coal samples under cyclic loading exhibits an obvious Kaiser Effect. The single-link cluster (SLC) method is applied to analyze the spatial evolution characteristics of AE events and the damage evolution process of coal samples. It is found that a subset scale of the SLC structure becomes smaller and smaller when the number of cyclic loading increases, and there is a negative linear relationship between the subset scale and the degree of damage. The spatial correlation length ξ of an SLC structure is calculated. The results show that ξ fluctuates around a certain value from the second cyclic loading process to the fifth cyclic loading process, but spatial correlation length ξ clearly increases in the sixth loading process. Based on the criterion of microcrack density, the coal sample failure process is the transformation from small-scale damage to large-scale damage, which is the reason for changes in the spatial correlation length. Through a systematic analysis, the SLC method is an effective method to research the damage evolution process of coal samples under cyclic loading, and will provide important reference values for studying coal bursts.

Purposeful Sampling for Qualitative Data Collection and Analysis in Mixed Method Implementation Research.

PubMed

Palinkas, Lawrence A; Horwitz, Sarah M; Green, Carla A; Wisdom, Jennifer P; Duan, Naihua; Hoagwood, Kimberly

2015-09-01

Purposeful sampling is widely used in qualitative research for the identification and selection of information-rich cases related to the phenomenon of interest. Although there are several different purposeful sampling strategies, criterion sampling appears to be used most commonly in implementation research. However, combining sampling strategies may be more appropriate to the aims of implementation research and more consistent with recent developments in quantitative methods. This paper reviews the principles and practice of purposeful sampling in implementation research, summarizes types and categories of purposeful sampling strategies and provides a set of recommendations for use of single strategy or multistage strategy designs, particularly for state implementation research.

Micromagnetic Cancer Cell Immobilization and Release for Real-Time Single Cell Analysis

NASA Astrophysics Data System (ADS)

Jaiswal, Devina; Rad, Armin Tahmasbi; Nieh, Mu-Ping; Claffey, Kevin P.; Hoshino, Kazunori

2017-04-01

Understanding the interaction of live cells with macromolecules is crucial for designing efficient therapies. Considering the functional heterogeneity found in cancer cells, real-time single cell analysis is necessary to characterize responses. In this study, we have designed and fabricated a microfluidic channel with patterned micromagnets which can temporarily immobilize the cells during analysis and release them after measurements. The microchannel is composed of plain coverslip top and bottom panels to facilitate easy microscopic observation and undisturbed application of analytes to the cells. Cells labeled with functionalized magnetic beads were immobilized in the device with an efficiency of 90.8±3.6%. Since the micromagnets are made of soft magnetic material (Ni), they released cells when external magnetic field was turned off from the channel. This allows the reuse of the channel for a new sample. As a model drug analysis, the immobilized breast cancer cells (MCF7) were exposed to fluorescent lipid nanoparticles and association and dissociation were measured through fluorescence analysis. Two concentrations of nanoparticles, 0.06 μg/ml and 0.08 μg/ml were tested and time lapse images were recorded and analyzed. The microfluidic device was able to provide a microenvironment for sample analysis, making it an efficient platform for real-time analysis.

Combining Raman and laser induced breakdown spectroscopy by double pulse lasing.

PubMed

Lednev, Vasily N; Pershin, Sergey M; Sdvizhenskii, Pavel A; Grishin, Mikhail Ya; Fedorov, Alexander N; Bukin, Vladimir V; Oshurko, Vadim B; Shchegolikhin, Alexander N

2018-01-01

A new approach combining Raman spectrometry and laser induced breakdown spectrometry (LIBS) within a single laser event was suggested. A pulsed solid state Nd:YAG laser running in double pulse mode (two frequency-doubled sequential nanosecond laser pulses with dozens microseconds delay) was used to combine two spectrometry methods within a single instrument (Raman/LIBS spectrometer). First, a low-energy laser pulse (power density far below ablation threshold) was used for Raman measurements while a second powerful laser pulse created the plasma suitable for LIBS analysis. A short time delay between two successive pulses allows measuring LIBS and Raman spectra at different moments but within a single laser flash-lamp pumping. Principal advantages of the developed instrument include high quality Raman/LIBS spectra acquisition (due to optimal gating for Raman/LIBS independently) and absence of target thermal alteration during Raman measurements. A series of high quality Raman and LIBS spectra were acquired for inorganic salts (gypsum, anhydrite) as well as for pharmaceutical samples (acetylsalicylic acid). To the best of our knowledge, the quantitative analysis feasibility by combined Raman/LIBS instrument was demonstrated for the first time by calibration curves construction for acetylsalicylic acid (Raman) and copper (LIBS) in gypsum matrix. Combining ablation pulses and Raman measurements (LIBS/Raman measurements) within a single instrument makes it an efficient tool for identification of samples hidden by non-transparent covering or performing depth profiling analysis including remote sensing. Graphical abstract Combining Raman and laser induced breakdown spectroscopy by double pulse lasing.

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities

PubMed Central

Leung, Kaston; Zahn, Hans; Leaver, Timothy; Konwar, Kishori M.; Hanson, Niels W.; Pagé, Antoine P.; Lo, Chien-Chi; Chain, Patrick S.; Hallam, Steven J.; Hansen, Carl L.

2012-01-01

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by “flow-controlled wetting,” a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members. PMID:22547789

Single-cell multimodal profiling reveals cellular epigenetic heterogeneity.

PubMed

Cheow, Lih Feng; Courtois, Elise T; Tan, Yuliana; Viswanathan, Ramya; Xing, Qiaorui; Tan, Rui Zhen; Tan, Daniel S W; Robson, Paul; Loh, Yuin-Han; Quake, Stephen R; Burkholder, William F

2016-10-01

Sample heterogeneity often masks DNA methylation signatures in subpopulations of cells. Here, we present a method to genotype single cells while simultaneously interrogating gene expression and DNA methylation at multiple loci. We used this targeted multimodal approach, implemented on an automated, high-throughput microfluidic platform, to assess primary lung adenocarcinomas and human fibroblasts undergoing reprogramming by profiling epigenetic variation among cell types identified through genotyping and transcriptional analysis.

ACVP-05: Virus Genetic Analysis from Cell-Free Plasma, Virally Infected Cells or Tissues and Cultured Supernatant Via Single Genome Amplification and Direct Sequencing | Frederick National Laboratory for Cancer Research

Cancer.gov

The Viral Evolution Core within the AIDS and Cancer Virus Program will extract viral RNA/DNA from cell-free or cell-associated samples. Complementary (cDNA) will be generated as needed, and cDNA or DNA will be diluted to a single copy prior to nested

Formability analysis of sheet metals by cruciform testing

NASA Astrophysics Data System (ADS)

Güler, B.; Alkan, K.; Efe, M.

2017-09-01

Cruciform biaxial tests are increasingly becoming popular for testing the formability of sheet metals as they achieve frictionless, in-plane, multi-axial stress states with a single sample geometry. However, premature fracture of the samples during testing prevents large strain deformation necessary for the formability analysis. In this work, we introduce a miniature cruciform sample design (few mm test region) and a test setup to achieve centre fracture and large uniform strains. With its excellent surface finish and optimized geometry, the sample deforms with diagonal strain bands intersecting at the test region. These bands prevent local necking and concentrate the strains at the sample centre. Imaging and strain analysis during testing confirm the uniform strain distributions and the centre fracture are possible for various strain paths ranging from plane-strain to equibiaxial tension. Moreover, the sample deforms without deviating from the predetermined strain ratio at all test conditions, allowing formability analysis under large strains. We demonstrate these features of the cruciform test for three sample materials: Aluminium 6061-T6 alloy, DC-04 steel and Magnesium AZ31 alloy, and investigate their formability at both the millimetre scale and the microstructure scale.

Isolation of Mitochondrial DNA from Single, Short Hairs without Roots Using Pressure Cycling Technology.

PubMed

Harper, Kathryn A; Meiklejohn, Kelly A; Merritt, Richard T; Walker, Jessica; Fisher, Constance L; Robertson, James M

2018-02-01

Hairs are commonly submitted as evidence to forensic laboratories, but standard nuclear DNA analysis is not always possible. Mitochondria (mt) provide another source of genetic material; however, manual isolation is laborious. In a proof-of-concept study, we assessed pressure cycling technology (PCT; an automated approach that subjects samples to varying cycles of high and low pressure) for extracting mtDNA from single, short hairs without roots. Using three microscopically similar donors, we determined the ideal PCT conditions and compared those yields to those obtained using the traditional manual micro-tissue grinder method. Higher yields were recovered from grinder extracts, but yields from PCT extracts exceeded the requirements for forensic analysis, with the DNA quality confirmed through sequencing. Automated extraction of mtDNA from hairs without roots using PCT could be useful for forensic laboratories processing numerous samples.

Wavelength-normalized spectroscopic analysis of Staphylococcus aureus and Pseudomonas aeruginosa growth rates.

PubMed

McBirney, Samantha E; Trinh, Kristy; Wong-Beringer, Annie; Armani, Andrea M

2016-10-01

Optical density (OD) measurements are the standard approach used in microbiology for characterizing bacteria concentrations in culture media. OD is based on measuring the optical absorbance of a sample at a single wavelength, and any error will propagate through all calculations, leading to reproducibility issues. Here, we use the conventional OD technique to measure the growth rates of two different species of bacteria, Pseudomonas aeruginosa and Staphylococcus aureus. The same samples are also analyzed over the entire UV-Vis wavelength spectrum, allowing a distinctly different strategy for data analysis to be performed. Specifically, instead of only analyzing a single wavelength, a multi-wavelength normalization process is implemented. When the OD method is used, the detected signal does not follow the log growth curve. In contrast, the multi-wavelength normalization process minimizes the impact of bacteria byproducts and environmental noise on the signal, thereby accurately quantifying growth rates with high fidelity at low concentrations.

Genetic Diversity among Bacillus anthracis Soil Isolates at Fine Geographic Scales

PubMed Central

Bader, Douglas E.

2012-01-01

Environmental samples were collected from carcass sites during and after anthrax outbreaks in 2000 and 2001 in the bison (Bison bison) population within Wood Buffalo National Park and the Hook Lake Region north of Wood Buffalo National Park. Bacillus anthracis spores were isolated from these samples and confirmed using phenotypic characterization and real-time PCR. Confirmed B. anthracis isolates were typed using multiple-locus variable-number tandem repeat analysis (MLVA15) and single-nucleotide-repeat analysis (SNRA). B. anthracis isolates split into two clades based on MLVA15, while SNRA allowed some isolates between carcass sites to be distinguished from each other. SNRA polymorphisms were also present within a single carcass site. Some isolates from different carcass sites having the same SNRA type had divergent MLVA types; this finding leads to questions about hierarchical typing methods and the robustness of the fine-scale typing of Bacillus anthracis. PMID:22773624

Repeatability and reproducibility of intracellular molar concentration assessed by synchrotron-based x-ray fluorescence microscopy

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

Merolle, L., E-mail: lucia.merolle@elettra.eu; Gianoncelli, A.; Malucelli, E., E-mail: emil.malucelli@unibo.it

2016-01-28

Elemental analysis of biological sample can give information about content and distribution of elements essential for human life or trace elements whose absence is the cause of abnormal biological function or development. However, biological systems contain an ensemble of cells with heterogeneous chemistry and elemental content; therefore, accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. Powerful methods in molecular biology are abundant, among them X-Ray microscopy based on synchrotron light source has gaining increasing attention thanks to its extremely sensitivity. However, reproducibility and repeatability of these measurements is one of the majormore » obstacles in achieving a statistical significance in single cells population analysis. In this study, we compared the elemental content of human colon adenocarcinoma cells obtained by three distinct accesses to synchrotron radiation light.« less

Prospects for dating monazite via single-collector HR-ICP-MS

NASA Astrophysics Data System (ADS)

Kohn, M. J.; Vervoort, J. D.

2006-12-01

ICP-MS analysis permits rapid and precise dating of minerals with high U and Th contents. Here we describe a new method for in situ determination of 206Pb/238U, 207Pb/^{235}U, ^{208}Pb/232Th, and 207Pb/206Pb ages in monazite via laser ablation (New Wave Research UP-213 laser system), single-collector, magnetic sector ICP-MS (ThermoFinnigan Element2), using spot sizes of 8-30 μm, a repetition rate of 5 Hz, and a fluence of 10 J/cm2. Based on analysis of 9 monazite samples of known ages ranging from 280 to 1800 Ma, analytical precision (single sample) is ±2-3% (2σ), and reproducibility (single sample) is ±2-4% (2σ), yielding age precisions of ±3- 5% (2σ) for single points, or ±1-2% (2 s.e.) for pooled multiple analyses (n > 4). Issues of accuracy are paramount. 207Pb/206Pb ages are consistently the most accurate and agree to ±2% with accepted TIMS ages. In contrast, 206Pb/238U, 207Pb/^{235}U, and ^{208}Pb/232Th ages can differ by as much as ±5% (2σ), a problem that has also been observed for SIMS Th-Pb dating. The sources of the interelement standardization disparities among monazites remain enigmatic, but do not result from molecular interferences on Pb, U, or Th peaks. Unresolvable mass interference between 204Pb and trace contaminant 204Hg in commercial Ar gas precludes precise common Pb corrections. Instead common Pb corrections are made assuming concordancy between 207Pb/^{235}U and either 206Pb/238U or ^{208}Pb/232Th ages. The new method offers rapid analysis (~1 minute), minimal sample preparation (polished thin section), and high sensitivity. Comparatively large errors on the 206Pb/238U, 207Pb/^{235}U, and ^{208}Pb/232Th ages will likely restrict analysis of younger monazite grains (<250 Ma) to applications where 5% accuracy is sufficient. Older grains (c. 500 Ma and older) can be dated more precisely and accurately using 207Pb/206Pb. One application to young materials involves dating a large vein monazite from the Llallagua tin district of Bolivia, which resolves a ~2 Myr history of mineralization at 20-22 Ma. These data support mineralization age estimates of 21 Ma (K-Ar on wallrock minerals) rather than 44 Ma (Sm-Nd on apatite).

GoIFISH: a system for the quantification of single cell heterogeneity from IFISH images.

PubMed

Trinh, Anne; Rye, Inga H; Almendro, Vanessa; Helland, Aslaug; Russnes, Hege G; Markowetz, Florian

2014-08-26

Molecular analysis has revealed extensive intra-tumor heterogeneity in human cancer samples, but cannot identify cell-to-cell variations within the tissue microenvironment. In contrast, in situ analysis can identify genetic aberrations in phenotypically defined cell subpopulations while preserving tissue-context specificity. GoIFISHGoIFISH is a widely applicable, user-friendly system tailored for the objective and semi-automated visualization, detection and quantification of genomic alterations and protein expression obtained from fluorescence in situ analysis. In a sample set of HER2-positive breast cancers GoIFISHGoIFISH is highly robust in visual analysis and its accuracy compares favorably to other leading image analysis methods. GoIFISHGoIFISH is freely available at www.sourceforge.net/projects/goifish/.

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

PubMed

Preissl, Sebastian; Fang, Rongxin; Huang, Hui; Zhao, Yuan; Raviram, Ramya; Gorkin, David U; Zhang, Yanxiao; Sos, Brandon C; Afzal, Veena; Dickel, Diane E; Kuan, Samantha; Visel, Axel; Pennacchio, Len A; Zhang, Kun; Ren, Bing

2018-03-01

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

PubMed

Mitsui, Jun; Fukuda, Yoko; Azuma, Kyo; Tozaki, Hirokazu; Ishiura, Hiroyuki; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

2010-07-01

We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

Evaluation of trace elements in lung samples from coal miners using neutron activation analysis.

PubMed

Saiki, M; Saldiva, P H; Alice, S H

1999-01-01

In this study, instrumental neutron activation analysis was applied to the determination of Sc, La, Ce, Nd, Sm, Eu, Tb, Yb, Lu, Hf, Th, and U in lung samples from miners working in coal mines located in the state of Santa Catarina, Brazil. These results were compared to those from a control group constituted of healthy individuals. The results showed that the elements determined exhibit considerable intersubject variability within a single group of individuals and the mean values of concentrations in miners' lungs were higher than those of normal individuals. Lung samples presented U concentrations varying from 11 to 890 micrograms/kg. Therefore, for some samples, the contribution of the uranium fission products in the analysis of La, Ce, Nd, and Sm was considered by determining the interference correction factors. The accuracy of the results was evaluated by analyzing certified reference materials.

Analysis of pollen load based on color, physicochemical composition and botanical source.

PubMed

Modro, Anna F H; Silva, Izabel C; Luz, Cynthia F P; Message, Dejair

2009-06-01

Pollen load samples from 10 hives of Apis mellifera (L.) were analyzed based on their physicochemical composition and botanical source, considering color as a parameter for quality control. In seven samples it was possible to establish the occurrence of more than 80% of a single pollen type, characterizing them as unifloral but with protein content variation. One of the samples was exclusively composed of saprophytic fungi (Cladosporium sp.). Comparing the mean results of the fungi loads with those of the nutritional components of pollen load, the former presented higher protein, mineral matter and dry matter and lower organic matter, ethereal extract and total carbohydrate values. The monochromatic samples met the physicochemical specifications regulating pollen load quality. The results showed that homogeneous coloration of the pollen load was not found to be a good indication of unifloral pollen, confirming the importance of physicochemical analysis and melissopalynological analysis for characterization of the quality of commercial pollen load.

Capillary absorption spectrometer and process for isotopic analysis of small samples

DOEpatents

Alexander, M. Lizabeth; Kelly, James F.; Sams, Robert L.; Moran, James J.; Newburn, Matthew K.; Blake, Thomas A.

2016-03-29

A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The method also permits sampling in vivo permitting real-time ambient studies of microbial communities.

Capillary absorption spectrometer and process for isotopic analysis of small samples

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

Alexander, M. Lizabeth; Kelly, James F.; Sams, Robert L.

A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The process also permits sampling in vivo permitting real-time ambient studies of microbial communities.

Sample injection and electrophoretic separation on a simple laminated paper based analytical device.

PubMed

Xu, Chunxiu; Zhong, Minghua; Cai, Longfei; Zheng, Qingyu; Zhang, Xiaojun

2016-02-01

We described a strategy to perform multistep operations on a simple laminated paper-based separation device by using electrokinetic flow to manipulate the fluids. A laminated crossed-channel paper-based separation device was fabricated by cutting a filter paper sheet followed by lamination. Multiple function units including sample loading, sample injection, and electrophoretic separation were integrated on a single paper based analytical device for the first time, by applying potential at different reservoirs for sample, sample waste, buffer, and buffer waste. As a proof-of-concept demonstration, mixed sample solution containing carmine and sunset yellow were loaded in the sampling channel, and then injected into separation channel followed by electrophoretic separation, by adjusting the potentials applied at the four terminals of sampling and separation channel. The effects of buffer pH, buffer concentration, channel width, and separation time on resolution of electrophoretic separation were studied. This strategy may be used to perform multistep operations such as reagent dilution, sample injection, mixing, reaction, and separation on a single microfluidic paper based analytical device, which is very attractive for building micro total analysis systems on microfluidic paper based analytical devices. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tunable Single-Cell Extraction for Molecular Analyses.

PubMed

Guillaume-Gentil, Orane; Grindberg, Rashel V; Kooger, Romain; Dorwling-Carter, Livie; Martinez, Vincent; Ossola, Dario; Pilhofer, Martin; Zambelli, Tomaso; Vorholt, Julia A

2016-07-14

Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level. Copyright © 2016 Elsevier Inc. All rights reserved.

Parallel Analysis with Unidimensional Binary Data

ERIC Educational Resources Information Center

Weng, Li-Jen; Cheng, Chung-Ping

2005-01-01

The present simulation investigated the performance of parallel analysis for unidimensional binary data. Single-factor models with 8 and 20 indicators were examined, and sample size (50, 100, 200, 500, and 1,000), factor loading (.45, .70, and .90), response ratio on two categories (50/50, 60/40, 70/30, 80/20, and 90/10), and types of correlation…

Integrated dual-tomography for refractive index analysis of free-floating single living cell with isotropic superresolution.

PubMed

B, Vinoth; Lai, Xin-Ji; Lin, Yu-Chih; Tu, Han-Yen; Cheng, Chau-Jern

2018-04-13

Digital holographic microtomography is a promising technique for three-dimensional (3D) measurement of the refractive index (RI) profiles of biological specimens. Measurement of the RI distribution of a free-floating single living cell with an isotropic superresolution had not previously been accomplished. To the best of our knowledge, this is the first study focusing on the development of an integrated dual-tomographic (IDT) imaging system for RI measurement of an unlabelled free-floating single living cell with an isotropic superresolution by combining the spatial frequencies of full-angle specimen rotation with those of beam rotation. A novel 'UFO' (unidentified flying object) like shaped coherent transfer function is obtained. The IDT imaging system does not require any complex image-processing algorithm for 3D reconstruction. The working principle was successfully demonstrated and a 3D RI profile of a single living cell, Candida rugosa, was obtained with an isotropic superresolution. This technology is expected to set a benchmark for free-floating single live sample measurements without labeling or any special sample preparations for the experiments.

The Use and Effectiveness of Triple Multiplex System for Coding Region Single Nucleotide Polymorphism in Mitochondrial DNA Typing of Archaeologically Obtained Human Skeletons from Premodern Joseon Tombs of Korea

PubMed Central

Oh, Chang Seok; Lee, Soong Deok; Kim, Yi-Suk; Shin, Dong Hoon

2015-01-01

Previous study showed that East Asian mtDNA haplogroups, especially those of Koreans, could be successfully assigned by the coupled use of analyses on coding region SNP markers and control region mutation motifs. In this study, we tried to see if the same triple multiplex analysis for coding regions SNPs could be also applicable to ancient samples from East Asia as the complementation for sequence analysis of mtDNA control region. By the study on Joseon skeleton samples, we know that mtDNA haplogroup determined by coding region SNP markers successfully falls within the same haplogroup that sequence analysis on control region can assign. Considering that ancient samples in previous studies make no small number of errors in control region mtDNA sequencing, coding region SNP analysis can be used as good complimentary to the conventional haplogroup determination, especially of archaeological human bone samples buried underground over long periods. PMID:26345190

Nanomanipulation-Coupled Matrix-Assisted Laser Desorption/ Ionization-Direct Organelle Mass Spectrometry: A Technique for the Detailed Analysis of Single Organelles

NASA Astrophysics Data System (ADS)

Phelps, Mandy S.; Sturtevant, Drew; Chapman, Kent D.; Verbeck, Guido F.

2016-02-01

We describe a novel technique combining precise organelle microextraction with deposition and matrix-assisted laser desorption/ionization (MALDI) for a rapid, minimally invasive mass spectrometry (MS) analysis of single organelles from living cells. A dual-positioner nanomanipulator workstation was utilized for both extraction of organelle content and precise co-deposition of analyte and matrix solution for MALDI-direct organelle mass spectrometry (DOMS) analysis. Here, the triacylglycerol (TAG) profiles of single lipid droplets from 3T3-L1 adipocytes were acquired and results validated with nanoelectrospray ionization (NSI) MS. The results demonstrate the utility of the MALDI-DOMS technique as it enabled longer mass analysis time, higher ionization efficiency, MS imaging of the co-deposited spot, and subsequent MS/MS capabilities of localized lipid content in comparison to NSI-DOMS. This method provides selective organellar resolution, which complements current biochemical analyses and prompts for subsequent subcellular studies to be performed where limited samples and analyte volume are of concern.

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

Vasdekis, Andreas E.; Stephanopoulos, Gregory

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

PubMed Central

Halpern, Micah D.; Molins, Claudia R.; Schriefer, Martin

2014-01-01

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi. PMID:24899074

Amphetamine concentrations in human urine following single-dose administration of the calcium antagonist prenylamine-studies using fluorescence polarization immunoassay (FPIA) and GC-MS.

PubMed

Kraemer, Thomas; Roditis, Susanne K; Peters, Frank T; Maurer, Hans H

2003-03-01

Prenylamine (R,S-N-(3,3-diphenylpropyl-methyl-2-phenethylamine), a World Health Organization class V calcium antagonist, is known to be metabolized to amphetamine. In this study, amphetamine concentrations after a single-dose administration of prenylamine were determined to check if they reached values that could be of analytical and/or pharmacological importance in clinical and forensic toxicology. Enantiomeric composition of amphetamine was also studied. Five volunteers received a single 120-mg oral dose of prenylamine. Urine samples were analyzed using the Abbott TDx immunoassay Amphetamine/Methamphetamine II and using our routine systematic toxicological analysis (STA) gas chromatography-mass spectrometry (GC-MS) procedure. For quantitation purposes, GC-MS was used in the selected-ion monitoring (SIM) mode (ions m/z 118, 122, 240, 244) after solid-phase extraction (Isolute Confirm HCX) and derivatization (heptafluorobutyric anhydride). Amphetamine-d5 was used as internal standard (IS). Chiral separation of the heptafluorobutyrated amphetamine enantiomers was achieved using an Astec Chiraldex G-PN column. The TDx results showed a great variability for the different volunteers. A urine sample of one volunteer showed results as high as 3200 ng/mL, whereas the urine samples of another volunteer never gave results greater than the TDx detection limit (100 ng/mL). Using the STA procedure, the presence of amphetamine could be confirmed in all urine samples with TDx results greater than the cutoff value (300 ng/mL). Using the GC-MS SIM method, amphetamine concentrations up to 1280 ng/mL were determined. Chiral analysis revealed that both enantiomers of amphetamine were present in the samples with a surplus of the S(+)-enantiomer in the early phase of excretion. Forensic implications are discussed.

The low single nucleotide polymorphism heritability of plasma and saliva cortisol levels.

PubMed

Neumann, Alexander; Direk, Nese; Crawford, Andrew A; Mirza, Saira; Adams, Hieab; Bolton, Jennifer; Hayward, Caroline; Strachan, David P; Payne, Erin K; Smith, Jennifer A; Milaneschi, Yuri; Penninx, Brenda; Hottenga, Jouke J; de Geus, Eco; Oldehinkel, Albertine J; van der Most, Peter J; de Rijke, Yolanda; Walker, Brian R; Tiemeier, Henning

2017-11-01

Cortisol is an important stress hormone affected by a variety of biological and environmental factors, such as the circadian rhythm, exercise and psychological stress. Cortisol is mostly measured using blood or saliva samples. A number of genetic variants have been found to contribute to cortisol levels with these methods. While the effects of several specific single genetic variants is known, the joint genome-wide contribution to cortisol levels is unclear. Our aim was to estimate the amount of cortisol variance explained by common single nucleotide polymorphisms, i.e. the SNP heritability, using a variety of cortisol measures, cohorts and analysis approaches. We analyzed morning plasma (n=5705) and saliva levels (n=1717), as well as diurnal saliva levels (n=1541), in the Rotterdam Study using genomic restricted maximum likelihood estimation. Additionally, linkage disequilibrium score regression was fitted on the results of genome-wide association studies (GWAS) performed by the CORNET consortium on morning plasma cortisol (n=12,597) and saliva cortisol (n=7703). No significant SNP heritability was detected for any cortisol measure, sample or analysis approach. Point estimates ranged from 0% to 9%. Morning plasma cortisol in the CORNET cohorts, the sample with the most power, had a 6% [95%CI: 0-13%] SNP heritability. The results consistently suggest a low SNP heritability of these acute and short-term measures of cortisol. The low SNP heritability may reflect the substantial environmental and, in particular, situational component of these cortisol measures. Future GWAS will require very large sample sizes. Alternatively, more long-term cortisol measures such as hair cortisol samples are needed to discover further genetic pathways regulating cortisol concentrations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization Data Package for Containerized Sludge Samples Collected from Engineered Container SCS-CON-210

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

Fountain, Matthew S.; Fiskum, Sandra K.; Baldwin, David L.

This data package contains the K Basin sludge characterization results obtained by Pacific Northwest National Laboratory during processing and analysis of four sludge core samples collected from Engineered Container SCS-CON-210 in 2010 as requested by CH2M Hill Plateau Remediation Company. Sample processing requirements, analytes of interest, detection limits, and quality control sample requirements are defined in the KBC-33786, Rev. 2. The core processing scope included reconstitution of a sludge core sample distributed among four to six 4-L polypropylene bottles into a single container. The reconstituted core sample was then mixed and subsampled to support a variety of characterization activities. Additionalmore » core sludge subsamples were combined to prepare a container composite. The container composite was fractionated by wet sieving through a 2,000 micron mesh and a 500-micron mesh sieve. Each sieve fraction was sampled to support a suite of analyses. The core composite analysis scope included density determination, radioisotope analysis, and metals analysis, including the Waste Isolation Pilot Plant Hazardous Waste Facility Permit metals (with the exception of mercury). The container composite analysis included most of the core composite analysis scope plus particle size distribution, particle density, rheology, and crystalline phase identification. A summary of the received samples, core sample reconstitution and subsampling activities, container composite preparation and subsampling activities, physical properties, and analytical results are presented. Supporting data and documentation are provided in the appendices. There were no cases of sample or data loss and all of the available samples and data are reported as required by the Quality Assurance Project Plan/Sampling and Analysis Plan.« less

Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting.

PubMed

Lamb, D C; Müller, B K; Bräuchle, C

2005-10-01

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.

Using Public Data for Comparative Proteome Analysis in Precision Medicine Programs.

PubMed

Hughes, Christopher S; Morin, Gregg B

2018-03-01

Maximizing the clinical utility of information obtained in longitudinal precision medicine programs would benefit from robust comparative analyses to known information to assess biological features of patient material toward identifying the underlying features driving their disease phenotype. Herein, the potential for utilizing publically deposited mass-spectrometry-based proteomics data to perform inter-study comparisons of cell-line or tumor-tissue materials is investigated. To investigate the robustness of comparison between MS-based proteomics studies carried out with different methodologies, deposited data representative of label-free (MS1) and isobaric tagging (MS2 and MS3 quantification) are utilized. In-depth quantitative proteomics data acquired from analysis of ovarian cancer cell lines revealed the robust recapitulation of observable gene expression dynamics between individual studies carried out using significantly different methodologies. The observed signatures enable robust inter-study clustering of cell line samples. In addition, the ability to classify and cluster tumor samples based on observed gene expression trends when using a single patient sample is established. With this analysis, relevant gene expression dynamics are obtained from a single patient tumor, in the context of a precision medicine analysis, by leveraging a large cohort of repository data as a comparator. Together, these data establish the potential for state-of-the-art MS-based proteomics data to serve as resources for robust comparative analyses in precision medicine applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Association of single nucleotide polymorphism in CD28(C/T-I3 + 17) and CD40 (C/T-1) genes with the Graves' disease.

PubMed

Mustafa, Saima; Fatima, Hira; Fatima, Sadia; Khosa, Tafheem; Akbar, Atif; Shaikh, Rehan Sadiq; Iqbal, Furhan

2018-01-01

To find out a correlation between the single nucleotide polymorphisms in cluster of differentiation 28 and cluster of differentiation 40 genes with Graves' disease, if any. This case-control study was conducted at the Multan Institute of Nuclear Medicine and Radiotherapy, Multan, Pakistan, and comprised blood samples of Graves' disease patients and controls. Various risk factors were also correlated either with the genotype at each single-nucleotide polymorphism or with various combinations of genotypes studied during present investigation. Of the 160 samples, there were 80(50%) each from patients and controls. Risk factor analysis revealed that gender (p=0.008), marital status (p<0.001), education (p<0.001), smoking (p<0.001), tri-iodothyronine (P <0.001), thyroxin (p<0.001) and thyroid-stimulating hormone (p<0.000) levels in blood were associated with Graves' disease. Both single-nucleotide polymorphisms in both genes were not associated with Graves' disease, either individually or in any combined form.

Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

NASA Astrophysics Data System (ADS)

Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

2016-06-01

Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10-100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

Imaging of Al/Fe ratios in synthetic Al-goethite revealed by nanoscale secondary ion mass spectrometry.

PubMed

Pohl, Lydia; Kölbl, Angelika; Werner, Florian; Mueller, Carsten W; Höschen, Carmen; Häusler, Werner; Kögel-Knabner, Ingrid

2018-04-30

Aluminium (Al)-substituted goethite is ubiquitous in soils and sediments. The extent of Al-substitution affects the physicochemical properties of the mineral and influences its macroscale properties. Bulk analysis only provides total Al/Fe ratios without providing information with respect to the Al-substitution of single minerals. Here, we demonstrate that nanoscale secondary ion mass spectrometry (NanoSIMS) enables the precise determination of Al-content in single minerals, while simultaneously visualising the variation of the Al/Fe ratio. Al-substituted goethite samples were synthesized with increasing Al concentrations of 0.1, 3, and 7 % and analysed by NanoSIMS in combination with established bulk spectroscopic methods (XRD, FTIR, Mössbauer spectroscopy). The high spatial resolution (50-150 nm) of NanoSIMS is accompanied by a high number of single-point measurements. We statistically evaluated the Al/Fe ratios derived from NanoSIMS, while maintaining the spatial information and reassigning it to its original localization. XRD analyses confirmed increasing concentration of incorporated Al within the goethite structure. Mössbauer spectroscopy revealed 11 % of the goethite samples generated at high Al concentrations consisted of hematite. The NanoSIMS data show that the Al/Fe ratios are in agreement with bulk data derived from total digestion and demonstrated small spatial variability between single-point measurements. More advantageously, statistical analysis and reassignment of single-point measurements allowed us to identify distinct spots with significantly higher or lower Al/Fe ratios. NanoSIMS measurements confirmed the capacity to produce images, which indicated the uniform increase in Al-concentrations in goethite. Using a combination of statistical analysis with information from complementary spectroscopic techniques (XRD, FTIR and Mössbauer spectroscopy) we were further able to reveal spots with lower Al/Fe ratios as hematite. Copyright © 2018 John Wiley & Sons, Ltd.

Alcoholic extraction enables EPR analysis to characterize radiation-induced cellulosic signals in spices.

PubMed

Ahn, Jae-Jun; Sanyal, Bhaskar; Akram, Kashif; Kwon, Joong-Ho

2014-11-19

Different spices such as turmeric, oregano, and cinnamon were γ-irradiated at 1 and 10 kGy. The electron paramagnetic resonance (EPR) spectra of the nonirradiated samples were characterized by a single central signal (g = 2.006), the intensity of which was significantly enhanced upon irradiation. The EPR spectra of the irradiated spice samples were characterized by an additional triplet signal at g = 2.006 with a hyperfine coupling constant of 3 mT, associated with the cellulose radical. EPR analysis on various sample pretreatments in the irradiated spice samples demonstrated that the spectral features of the cellulose radical varied on the basis of the pretreatment protocol. Alcoholic extraction pretreatment produced considerable improvements of the EPR signals of the irradiated spice samples relative to the conventional oven and freeze-drying techniques. The alcoholic extraction process is therefore proposed as the most suitable sample pretreatment for unambiguous detection of irradiated spices by EPR spectroscopy.

Capillary sample introduction of polymerase chain reaction (PCR) products separated in ultrathin slab gels.

PubMed

Bullard, K M; Hietpas, P B; Ewing, A G

1998-01-01

Polymerase chain reaction (PCR) amplified short tandem repeat (STR) samples from the HUMVWF locus have been analyzed using a unique sample introduction and separation technique. A single capillary is used to transfer samples onto an ultrathin slab gel (57 microm thin). This ultrathin nondenaturing polyacrylamide gel is used to separate the amplified fragments, and laser-induced fluorescence with ethidium bromide is used for detection. The feasibility of performing STR analysis using this system has been investigated by examining the reproducibility for repeated samples. Reproducibility is examined by comparing the migration of the 14 and 17 HUMVWF alleles on three consecutive separations on the ultrathin slab gel. Using one locus, separations match in migration time with the two alleles 42 s apart for each of the three consecutive separations. This technique shows potential to increase sample throughput in STR analysis techniques although separation resolution still needs to be improved.

PRIMUS/NAVCARE Cost-Effectiveness Analysis

DTIC Science & Technology

1991-04-08

ICD-9-CM diagnosis codes that occurred most frequently in the medical record sample - 328.9 ( otitis media , unspecified) and 465.9 (upper...when attention is focused upon a single diagnosis, the MTF CECs are no longer consistently above the PRIMUS CECs. For otitis media , the MTF CECs are...CHAMPUS-EQUIVALENT COSTS FOR SELECTED DIAGNOSES 328.9 OTITIS MEDIA , UNSPECIFIED Sample Size Mean 95% Confidence Interval Upper Limit Lower

Identification of Biomarkers Associated with the Healing of Chronic Wounds

DTIC Science & Technology

2015-11-01

The analysis of the wound fluid began with a broad survey tool Kinex™ Antibody Microarray (KAM) a single dye , non-competitive sample binding...signaling proteins. Lysate protein from each sample was covalently labeled with a fluorescent dye combination. Free dye molecules were then...patterned structures is controlled by varying their pattern geometry. The biodegradation of micro-patterned structures is modeled geometrically based on

The Structure of Self-Serving Cognitive Distortions: A Validation of the "How I Think" Questionnaire in a Sample of Italian Adolescents

ERIC Educational Resources Information Center

Bacchini, Dario; De Angelis, Grazia; Affuso, Gaetana; Brugman, Daniel

2016-01-01

This study aims to investigate the structure of self-serving cognitive distortions (CD), evaluating the psychometric properties of the How I Think Questionnaire in a sample of Italian adolescents. A confirmatory factor analysis supported the distinction between four categories of CD and the use of a single second-order dimension of CD. Reliability…

Ocular findings associated with a Cys39Arg mutation in the Norrie disease gene.

PubMed

Joos, K M; Kimura, A E; Vandenburgh, K; Bartley, J A; Stone, E M

1994-12-01

To diagnose the carriers and noncarriers in a family affected with Norrie disease based on molecular analysis. Family members from three generations, including one affected patient, two obligate carriers, one carrier identified with linkage analysis, one noncarrier identified with linkage analysis, and one female family member with indeterminate carrier status, were examined clinically and electrophysiologically. Linkage analysis had previously failed to determine the carrier status of one female family member in the third generation. Blood samples were screened for mutations in the Norrie disease gene with single-strand conformation polymorphism analysis. The mutation was characterized by dideoxy-termination sequencing. Ophthalmoscopy and electroretinographic examination failed to detect the carrier state. The affected individuals and carriers in this family were found to have a transition from thymidine to cytosine in the first nucleotide of codon 39 of the Norrie disease gene, causing a cysteine-to-arginine mutation. Single-strand conformation polymorphism analysis identified a patient of indeterminate status (by linkage) to be a noncarrier of Norrie disease. Ophthalmoscopy and electroretinography could not identify carriers of this Norrie disease mutation. Single-strand conformation polymorphism analysis was more sensitive and specific than linkage analysis in identifying carriers in this family.

Confirmatory factor analysis of the Autonomy over Tobacco Scale (AUTOS) in adults.

PubMed

Wellman, Robert J; DiFranza, Joseph R; O'Loughlin, Jennifer

2015-11-01

The Autonomy over Tobacco Scale (AUTOS), a 12-item self-administered questionnaire, was designed to measure autonomy in three correlated lower-order symptom domains: withdrawal, psychological dependence, and cue-induced craving. The factor structure of the AUTOS remains an open question; confirmatory analyses in adolescents supported the hierarchical structure, while exploratory analyses in adolescents and adults yield single-factor solutions. Here we seek to determine whether the hypothesized hierarchical structure is valid in adult smokers. The AUTOS was administered to two independent convenience samples of adult current smokers: a calibration sample recruited in the US for online studies, and a confirmation sample drawn from the prospective Nicotine Dependence in Teens study in Montreal. We tested competing hierarchical and single-factor models using the robust weighted least-squares (WLSMV) estimation method. A single-factor model that allowed correlated error variances between theoretically related items fit well in the calibration sample (n = 434), χ(2)SB(52) = 165.71; χ(2)/df = 3.19; SRMR = 0.03; CFI = 0.96; NNFI = 0.95; RMSEA = 0.07 (95% CI: 0.06, 0.08). Reliability of the single factor was high (ωB = 0.92) and construct validity was adequate. In the confirmation sample (n = 335), a similar model fit well:χ(2)SB(53) = 126.94; χ(2)/df = 2.44; SRMR = 0.04; CFI = 0.95; NNFI = 0.93; RMSEA = 0.07 (95% CI: 0.05, 0.08). Reliability of the single factor was again high (ωB = 0.92) and construct validity was adequate. The AUTOS is unidimensional in adult smokers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Experiments on Nucleation in Different Flow Regimes

NASA Technical Reports Server (NTRS)

Bayuzick, Robert J.

1999-01-01

The vast majority of metallic engineering materials are solidified from the liquid phase. Understanding the solidification process is essential to control microstructure, which in turn, determines the properties of materials. The genesis of solidification is nucleation, where the first stable solid forms from the liquid phase. Nucleation kinetics determine the degree of undercooling and phase selection. As such, it is important to understand nucleation phenomena in order to control solidification or glass formation in metals and alloys. Early experiments in nucleation kinetics were accomplished by droplet dispersion methods [1-6]. Dilitometry was used by Turnbull and others, and more recently differential thermal analysis and differential scanning calorimetry have been used for kinetic studies. These techniques have enjoyed success; however, there are difficulties with these experiments. Since materials are dispersed in a medium, the character of the emulsion/metal interface affects the nucleation behavior. Statistics are derived from the large number of particles observed in a single experiment, but dispersions have a finite size distribution which adds to the uncertainty of the kinetic determinations. Even though temperature can be controlled quite well before the onset of nucleation, the release of the latent heat of fusion during nucleation of particles complicates the assumption of isothermality during these experiments. Containerless processing has enabled another approach to the study of nucleation kinetics [7]. With levitation techniques it is possible to undercool one sample to nucleation repeatedly in a controlled manner, such that the statistics of the nucleation process can be derived from multiple experiments on a single sample. The authors have fully developed the analysis of nucleation experiments on single samples following the suggestions of Skripov [8]. The advantage of these experiments is that the samples are directly observable. The nucleation temperature can be measured by noncontact optical pyrometry, the mass of the sample is known, and post processing analysis can be conducted on the sample. The disadvantages are that temperature measurement must have exceptionally high precision, and it is not possible to isolate specific heterogeneous sites as in droplet dispersions.

Investigation of pyrolysis kinetics of humic acids from low rank Anatolian coal by thermal analysis

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

Tonbul, Y.; Erdogan, S.

2007-07-01

Thermogravimetric analysis (TGA) of humic acid samples from low rank Anatolian (east of Turkey, Bingol) coal were investigated under atmospheric pressure. The samples were subjected for the decomposition of organic matter ambient to 800{sup o} C at four different heating rates (5, 10, 15, and 20 degrees C min{sup -1}). The humic acid samples were started at decomposition between 170 - 206{sup o}C and amount of residues varied 55-60% according to heating rate. Each of samples showed a single step mass loss. TG/DTG data of samples were analyzed to determine activation energy values by Coats and Redfern method and Arrheniusmore » method. Activation energy values are similar obtained from Coats and Redfern method and Arrhenius method and varied from 25 to 29 kJ mol{sup -1}.« less

Preferential access to genetic information from endogenous hominin ancient DNA and accurate quantitative SNP-typing via SPEX

PubMed Central

Brotherton, Paul; Sanchez, Juan J.; Cooper, Alan; Endicott, Phillip

2010-01-01

The analysis of targeted genetic loci from ancient, forensic and clinical samples is usually built upon polymerase chain reaction (PCR)-generated sequence data. However, many studies have shown that PCR amplification from poor-quality DNA templates can create sequence artefacts at significant levels. With hominin (human and other hominid) samples, the pervasive presence of highly PCR-amplifiable human DNA contaminants in the vast majority of samples can lead to the creation of recombinant hybrids and other non-authentic artefacts. The resulting PCR-generated sequences can then be difficult, if not impossible, to authenticate. In contrast, single primer extension (SPEX)-based approaches can genotype single nucleotide polymorphisms from ancient fragments of DNA as accurately as modern DNA. A single SPEX-type assay can amplify just one of the duplex DNA strands at target loci and generate a multi-fold depth-of-coverage, with non-authentic recombinant hybrids reduced to undetectable levels. Crucially, SPEX-type approaches can preferentially access genetic information from damaged and degraded endogenous ancient DNA templates over modern human DNA contaminants. The development of SPEX-type assays offers the potential for highly accurate, quantitative genotyping from ancient hominin samples. PMID:19864251

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

PubMed

Spits, C; Le Caignec, C; De Rycke, M; Van Haute, L; Van Steirteghem, A; Liebaers, I; Sermon, K

2006-05-01

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research. (c) 2006 Wiley-Liss, Inc.

Single column comprehensive analysis of pharmaceutical preparations using dual-injection mixed-mode (ion-exchange and reversed-phase) and hydrophilic interaction liquid chromatography.

PubMed

Kazarian, Artaches A; Taylor, Mark R; Haddad, Paul R; Nesterenko, Pavel N; Paull, Brett

2013-12-01

The comprehensive separation and detection of hydrophobic and hydrophilic active pharmaceutical ingredients (APIs), their counter-ions (organic, inorganic) and excipients, using a single mixed-mode chromatographic column, and a dual injection approach is presented. Using a mixed-mode Thermo Fisher Acclaim Trinity P1 column, APIs, their counter-ions and possible degradants were first separated using a combination of anion-exchange, cation-exchange and hydrophobic interactions, using a mobile phase consisting of a dual organic modifier/salt concentration gradient. A complementary method was also developed using the same column for the separation of hydrophilic bulk excipients, using hydrophilic interaction liquid chromatography (HILIC) under high organic solvent mobile phase conditions. These two methods were then combined within a single gradient run using dual sample injection, with the first injection at the start of the applied gradient (mixed-mode retention of solutes), followed by a second sample injection at the end of the gradient (HILIC retention of solutes). Detection using both ultraviolet absorbance and refractive index enabled the sensitive detection of APIs and UV-absorbing counter-ions, together with quantitative determination of bulk excipients. The developed approach was applied successfully to the analysis of a dry powder inhalers (Flixotide(®), Spiriva(®)), enabling comprehensive quantification of all APIs and excipients in the sample. Copyright © 2013 Elsevier B.V. All rights reserved.

Tank characterization report for single-shell tank 241-U-110. Revision 1

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

Brown, T.M.; Jensen, L.

1993-09-01

Tank 241-U-110 (U-110) is a Hanford Site waste tank that was ;most recently sampled in November and December 1989. Analysis of the samples obtained from tank U-110 was conducted to support the characterization of the contents of this tank and to support Hanford Federal Facility Agreement and Consent Order milestone M-10-00 (Ecology, et al. 1992). Because of incomplete recovery of the waste during sampling, there may be bias in the results of this characterization report.

An Optimal Bahadur-Efficient Method in Detection of Sparse Signals with Applications to Pathway Analysis in Sequencing Association Studies.

PubMed

Dai, Hongying; Wu, Guodong; Wu, Michael; Zhi, Degui

2016-01-01

Next-generation sequencing data pose a severe curse of dimensionality, complicating traditional "single marker-single trait" analysis. We propose a two-stage combined p-value method for pathway analysis. The first stage is at the gene level, where we integrate effects within a gene using the Sequence Kernel Association Test (SKAT). The second stage is at the pathway level, where we perform a correlated Lancaster procedure to detect joint effects from multiple genes within a pathway. We show that the Lancaster procedure is optimal in Bahadur efficiency among all combined p-value methods. The Bahadur efficiency,[Formula: see text], compares sample sizes among different statistical tests when signals become sparse in sequencing data, i.e. ε →0. The optimal Bahadur efficiency ensures that the Lancaster procedure asymptotically requires a minimal sample size to detect sparse signals ([Formula: see text]). The Lancaster procedure can also be applied to meta-analysis. Extensive empirical assessments of exome sequencing data show that the proposed method outperforms Gene Set Enrichment Analysis (GSEA). We applied the competitive Lancaster procedure to meta-analysis data generated by the Global Lipids Genetics Consortium to identify pathways significantly associated with high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol.

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

PubMed

Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

2016-01-01

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh

PubMed Central

Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

2016-01-01

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country. PMID:27082248

Electrical, structural and thermal studies of carbon nanotubes from natural legume seeds: kala chana

NASA Astrophysics Data System (ADS)

Ranu, Rachana; Chauhan, Yatishwar; Singh, Pramod K.; Bhattacharya, B.; Tomar, S. K.

2016-12-01

Carbon nanotubes (CNTs) are the carbon materials measured at nanoscale level and they are defined in two types according to the number of concentric layers, i.e. single-layer tube is single-walled nanotubes, while multi-layer tube structure is called multi-walled nanotubes. The green method synthesis for the preparation of CNTs begins with the smashing of legume seeds kala chana, and then they form complex with cobalt salt. Desiccation of the complex compound forms cobalt salt and seed protein. The complex is then decomposed at 625 °C in muffle furnace for 20 min. Purification of the decomposed sample is done through acid wash treatment and dried in vacuum oven. The confirmations of CNTs are done by nuclear magnetic resonance and Fourier transform infrared, which analyzes the denatured protein, reacted to the metal salt. X-Ray diffraction determines the MWNTs with transmission electron microscope (TEM) reports the network structure of CNTs. thermal gravimetric analysis (TGA)-differential thermal analysis (DTA)-thermogravimetric analysis (DTG) tests the amount of sample under thermal treatment. Vibrating sample magnetometer determines the paramagnetic nature of CNTs. CNTs thus prepared can be used in mechanical fields, in solar cells, in electronics fields, etc. because of their multidisciplinary properties. The synthesized CNTs are eco-friendly in nature, prepared by the legume seed natural precursor.

Comprehensive two-dimensional liquid chromatography for polyphenol analysis in foodstuffs.

PubMed

Cacciola, Francesco; Farnetti, Sara; Dugo, Paola; Marriott, Philip John; Mondello, Luigi

2017-01-01

Polyphenols are a class of plant secondary metabolites that are recently drawing a special interest because of their broad spectrum of pharmacological effects. As they are characterized by an enormous structural variability, the identification of these molecules in food samples is a difficult task, and sometimes having only a limited number of commercially available reference materials is not of great help. One-dimensional liquid chromatography is the most widely applied analytical approach for their analysis. In particular, the hyphenation of liquid chromatography to mass spectrometry has come to play an influential role by allowing relatively fast tentative identification and accurate quantification of polyphenolic compounds at trace levels in vegetable media. However, when dealing with very complex real-world food samples, a single separation system often does not provide sufficient resolving power for attaining rewarding results. Comprehensive two-dimensional liquid chromatography is a technique of great analytical impact, since it offers much higher peak capacities than separations in a single dimension. In the present review, we describe applications in the field of comprehensive two-dimensional liquid chromatography for polyphenol analysis in real-world food samples. Comprehensive two-dimensional liquid chromatography applications to nonfood matrices fall outside the scope of the current report and will not be discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Growth, optical, ICP and thermal studies of nonlinear optical single crystal: Sodium acid phthalate (NaAP)

NASA Astrophysics Data System (ADS)

Mahadevan, M.; Arivanandhan, M.; Elangovan, K.; Anandan, P.; Ramachandran, K.

2017-07-01

Good quality single crystals of sodium acid phthalate (NaAP) were grown by slow evaporation technique. Single crystal X-ray diffraction study of the grown crystal reveals that the crystal belongs to orthorhombic system with space group B2ab. Fourier transform infrared spectrum confirms the presence of the functional groups of the grown material. Inductively coupled plasma emission spectroscopy analysis is used to confirm the presence of Na element in the sample. Thermal analysis of the NaAP crystal shows that the crystal is stable up to 140°C. Optical transmittance of the grown crystal was recorded in the wavelength range from 200 and 800 nm using UV-Vis-NIR spectrophotometer. The second harmonic generation of NaAP was analysed using Kurtz powder technique.

Quantitative gene expression analysis in Caenorhabditis elegans using single molecule RNA FISH.

PubMed

Bolková, Jitka; Lanctôt, Christian

2016-04-01

Advances in fluorescent probe design and synthesis have allowed the uniform in situ labeling of individual RNA molecules. In a technique referred to as single molecule RNA FISH (smRNA FISH), the labeled RNA molecules can be imaged as diffraction-limited spots and counted using image analysis algorithms. Single RNA counting has provided valuable insights into the process of gene regulation. This microscopy-based method has often revealed a high cell-to-cell variability in expression levels, which has in turn led to a growing interest in investigating the biological significance of gene expression noise. Here we describe the application of the smRNA FISH technique to samples of Caenorhabditis elegans, a well-characterized model organism. Copyright © 2015 Elsevier Inc. All rights reserved.

Short-Term Intra-Subject Variation in Exhaled Volatile Organic Compounds (VOCs) in COPD Patients and Healthy Controls and Its Effect on Disease Classification

PubMed Central

Phillips, Christopher; Mac Parthaláin, Neil; Syed, Yasir; Deganello, Davide; Claypole, Timothy; Lewis, Keir

2014-01-01

Exhaled volatile organic compounds (VOCs) are of interest for their potential to diagnose disease non-invasively. However, most breath VOC studies have analyzed single breath samples from an individual and assumed them to be wholly consistent representative of the person. This provided the motivation for an investigation of the variability of breath profiles when three breath samples are taken over a short time period (two minute intervals between samples) for 118 stable patients with Chronic Obstructive Pulmonary Disease (COPD) and 63 healthy controls and analyzed by gas chromatography and mass spectroscopy (GC/MS). The extent of the variation in VOC levels differed between COPD and healthy subjects and the patterns of variation differed for isoprene versus the bulk of other VOCs. In addition, machine learning approaches were applied to the breath data to establish whether these samples differed in their ability to discriminate COPD from healthy states and whether aggregation of multiple samples, into single data sets, could offer improved discrimination. The three breath samples gave similar classification accuracy to one another when evaluated separately (66.5% to 68.3% subjects classified correctly depending on the breath repetition used). Combining multiple breath samples into single data sets gave better discrimination (73.4% subjects classified correctly). Although accuracy is not sufficient for COPD diagnosis in a clinical setting, enhanced sampling and analysis may improve accuracy further. Variability in samples, and short-term effects of practice or exertion, need to be considered in any breath testing program to improve reliability and optimize discrimination. PMID:24957028

Short-Term Intra-Subject Variation in Exhaled Volatile Organic Compounds (VOCs) in COPD Patients and Healthy Controls and Its Effect on Disease Classification.

PubMed

Phillips, Christopher; Mac Parthaláin, Neil; Syed, Yasir; Deganello, Davide; Claypole, Timothy; Lewis, Keir

2014-05-09

Exhaled volatile organic compounds (VOCs) are of interest for their potential to diagnose disease non-invasively. However, most breath VOC studies have analyzed single breath samples from an individual and assumed them to be wholly consistent representative of the person. This provided the motivation for an investigation of the variability of breath profiles when three breath samples are taken over a short time period (two minute intervals between samples) for 118 stable patients with Chronic Obstructive Pulmonary Disease (COPD) and 63 healthy controls and analyzed by gas chromatography and mass spectroscopy (GC/MS). The extent of the variation in VOC levels differed between COPD and healthy subjects and the patterns of variation differed for isoprene versus the bulk of other VOCs. In addition, machine learning approaches were applied to the breath data to establish whether these samples differed in their ability to discriminate COPD from healthy states and whether aggregation of multiple samples, into single data sets, could offer improved discrimination. The three breath samples gave similar classification accuracy to one another when evaluated separately (66.5% to 68.3% subjects classified correctly depending on the breath repetition used). Combining multiple breath samples into single data sets gave better discrimination (73.4% subjects classified correctly). Although accuracy is not sufficient for COPD diagnosis in a clinical setting, enhanced sampling and analysis may improve accuracy further. Variability in samples, and short-term effects of practice or exertion, need to be considered in any breath testing program to improve reliability and optimize discrimination.

Delayed matching to two-picture samples by individuals with and without disabilities: an analysis of the role of naming.

PubMed

Gutowski, Stanley J; Stromer, Robert

2003-01-01

Delayed matching to complex, two-picture samples (e.g., cat-dog) may be improved when the samples occasion differential verbal behavior. In Experiment 1, individuals with mental retardation matched picture comparisons to identical single-picture samples or to two-picture samples, one of which was identical to a comparison. Accuracy scores were typically high on single-picture trials under both simultaneous and delayed matching conditions. Scores on two-picture trials were also high during the simultaneous condition but were lower during the delay condition. However, scores improved on delayed two-picture trials when each of the sample pictures was named aloud before comparison responding. Experiment 2 replicated these results with preschoolers with typical development and a youth with mental retardation. Sample naming also improved the preschoolers' matching when the samples were pairs of spoken names and the correct comparison picture matched one of the names. Collectively, the participants could produce the verbal behavior that might have improved performance, but typically did not do so unless the procedure required it. The success of the naming intervention recommends it for improving the observing and remembering of multiple elements of complex instructional stimuli.

Measurement of the Electroweak Single Top Quark Production Cross Section and the CKM Matrix Element $$|V_{tb}|$$ at CDF Run II

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

Larana, Bruno Casal

2010-01-01

The establishment of the electroweak single top quark production at CDF is experimentally challenging. The small single top signal hidden under large uncertain background processes makes it necessary an excellent understanding of the detector and a detailed study of the processes involved. Moreover, simple counting experiments are not sufficient to extract enough information from the candidate event sample and multivariate analysis techniques are crucial to distinguish signal from background. This thesis presents the world’s most sensitive individual search, together with CDF’s Neural Network analysis, for the combined s- and t-channel single top production. This analysis uses a dataset that correspondsmore » to an integrated luminosity of 3.2fb -1, and is based on a Boosted Decision Tree method that combines information from several input variables to construct a final powerful discriminant, reaching a sensitivity to the combined single top quark production equivalent to 5.2σ. The measured combined single top quark production cross section is 2.1 +0.7 -0.6 pb assuming a top quark mass of 175 GeV/c 2. The probability that this result comes from a background-only fluctuation (p-value) is 0.0002, which corresponds to 3.5σ.« less

High-throughput quantitative analysis by desorption electrospray ionization mass spectrometry.

PubMed

Manicke, Nicholas E; Kistler, Thomas; Ifa, Demian R; Cooks, R Graham; Ouyang, Zheng

2009-02-01

A newly developed high-throughput desorption electrospray ionization (DESI) source was characterized in terms of its performance in quantitative analysis. A 96-sample array, containing pharmaceuticals in various matrices, was analyzed in a single run with a total analysis time of 3 min. These solution-phase samples were examined from a hydrophobic PTFE ink printed on glass. The quantitative accuracy, precision, and limit of detection (LOD) were characterized. Chemical background-free samples of propranolol (PRN) with PRN-d(7) as internal standard (IS) and carbamazepine (CBZ) with CBZ-d(10) as IS were examined. So were two other sample sets consisting of PRN/PRN-d(7) at varying concentration in a biological milieu of 10% urine or porcine brain total lipid extract, total lipid concentration 250 ng/microL. The background-free samples, examined in a total analysis time of 1.5 s/sample, showed good quantitative accuracy and precision, with a relative error (RE) and relative standard deviation (RSD) generally less than 3% and 5%, respectively. The samples in urine and the lipid extract required a longer analysis time (2.5 s/sample) and showed RSD values of around 10% for the samples in urine and 4% for the lipid extract samples and RE values of less than 3% for both sets. The LOD for PRN and CBZ when analyzed without chemical background was 10 and 30 fmol, respectively. The LOD of PRN increased to 400 fmol analyzed in 10% urine, and 200 fmol when analyzed in the brain lipid extract.

A consensus least squares support vector regression (LS-SVR) for analysis of near-infrared spectra of plant samples.

PubMed

Li, Yankun; Shao, Xueguang; Cai, Wensheng

2007-04-15

Consensus modeling of combining the results of multiple independent models to produce a single prediction avoids the instability of single model. Based on the principle of consensus modeling, a consensus least squares support vector regression (LS-SVR) method for calibrating the near-infrared (NIR) spectra was proposed. In the proposed approach, NIR spectra of plant samples were firstly preprocessed using discrete wavelet transform (DWT) for filtering the spectral background and noise, then, consensus LS-SVR technique was used for building the calibration model. With an optimization of the parameters involved in the modeling, a satisfied model was achieved for predicting the content of reducing sugar in plant samples. The predicted results show that consensus LS-SVR model is more robust and reliable than the conventional partial least squares (PLS) and LS-SVR methods.

Multicomponent blood lipid analysis by means of near infrared spectroscopy, in geese.

PubMed

Bazar, George; Eles, Viktoria; Kovacs, Zoltan; Romvari, Robert; Szabo, Andras

2016-08-01

This study provides accurate near infrared (NIR) spectroscopic models on some laboratory determined clinicochemical parameters (i.e. total lipid (5.57±1.95 g/l), triglyceride (2.59±1.36 mmol/l), total cholesterol (3.81±0.68 mmol/l), high density lipoprotein (HDL) cholesterol (2.45±0.58 mmol/l)) of blood serum samples of fattened geese. To increase the performance of multivariate chemometrics, samples significantly deviating from the regression models implying laboratory error were excluded from the final calibration datasets. Reference data of excluded samples having outlier spectra in principal component analysis were not marked as false. Samples deviating from the regression models but having non outlier spectra in PCA were identified as having false reference constituent values. Based on the NIR selection methods, 5% of the reference measurement data were rated as doubtful. The achieved models reached R(2) of 0.864, 0.966, 0.850, 0.793, and RMSE of 0.639 g/l, 0.232 mmol/l, 0.210 mmol/l, 0.241 mmol/l for total lipid, triglyceride, total cholesterol and HDL cholesterol, respectively, during independent validation. Classical analytical techniques focus on single constituents and often require chemicals, time-consuming measurements, and experienced technicians. NIR technique provides a quick, cost effective, non-hazardous alternative method for analysis of several constituents based on one single spectrum of each sample, and it also offers the possibility for looking at the laboratory reference data critically. Evaluation of reference data to identify and exclude falsely analyzed samples can provide warning feedback to the reference laboratory, especially in the case of analyses where laboratory methods are not perfectly suited to the subjected material and there is an increased chance of laboratory error. Copyright © 2016 Elsevier B.V. All rights reserved.

Direct Surface and Droplet Microsampling for Electrospray Ionization Mass Spectrometry Analysis with an Integrated Dual-Probe Microfluidic Chip

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

Huang, Cong-Min; Zhu, Ying; Jin, Di-Qiong

Ambient mass spectrometry (MS) has revolutionized the way of MS analysis and broadened its application in various fields. This paper describes the use of microfluidic techniques to simplify the setup and improve the functions of ambient MS by integrating the sampling probe, electrospray emitter probe, and online mixer on a single glass microchip. Two types of sampling probes, including a parallel-channel probe and a U-shaped channel probe, were designed for dryspot and liquid-phase droplet samples, respectively. We demonstrated that the microfabrication techniques not only enhanced the capability of ambient MS methods in analysis of dry-spot samples on various surfaces, butmore » also enabled new applications in the analysis of nanoliter-scale chemical reactions in an array of droplets. The versatility of the microchip-based ambient MS method was demonstrated in multiple different applications including evaluation of residual pesticide on fruit surfaces, sensitive analysis of low-ionizable analytes using postsampling derivatization, and high-throughput screening of Ugi-type multicomponent reactions.« less

Carbon-hydrogen-related complexes in Si

NASA Astrophysics Data System (ADS)

Kolkovsky, Vl.; Stübner, R.; Gwozdz, K.; Weber, J.

2018-04-01

Several deep level transient spectroscopy (DLTS) peaks (E42, E65, E75, E90, E262, and H180) are observed in n- and p-type Czochralski-grown Si samples subjected to hydrogenation by a dc H plasma treatment. The concentration of the defects is found to be proportional to the carbon and hydrogen content in our samples. The analysis of the depth profiles performed in Si samples hydrogenated by wet chemically etching shows that all these defects contain a single H atom. E65 and E75 appear only in samples with a high oxygen content which shows that oxygen is a constituent of these defects. The analysis of the enhancement of the emission rate of the defects with electric field shows that E65, E75, E90, and E262 are single acceptors whereas E42 is a double acceptor. The presence of a barrier for hole capture (about 53 meV) can explain the absence of the enhancement of the emission rate of H180, which can be attributed to a single acceptor state. From a comparison with theory, we assign E90 to CH1BC, E42 (E262) to CH1AB, and H180 to CH1Td. The similarity of the electrical properties of E65 and E75 to those of E90 suggest that E65 and E75 may originate from the CH1BC defect with an oxygen atom in its nearest neighborhood. Our results on the CH-related complexes give a conclusive explanation of some previously reported controversial experimental data.

Multi-Genetic Marker Approach and Spatio-Temporal Analysis Suggest There Is a Single Panmictic Population of Swordfish Xiphias gladius in the Indian Ocean

PubMed Central

Muths, Delphine; Le Couls, Sarah; Evano, Hugues; Grewe, Peter; Bourjea, Jerome

2013-01-01

Genetic population structure of swordfish Xiphias gladius was examined based on 2231 individual samples, collected mainly between 2009 and 2010, among three major sampling areas within the Indian Ocean (IO; twelve distinct sites), Atlantic (two sites) and Pacific (one site) Oceans using analysis of nineteen microsatellite loci (n = 2146) and mitochondrial ND2 sequences (n = 2001) data. Sample collection was stratified in time and space in order to investigate the stability of the genetic structure observed with a special focus on the South West Indian Ocean. Significant AMOVA variance was observed for both markers indicating genetic population subdivision was present between oceans. Overall value of F-statistics for ND2 sequences confirmed that Atlantic and Indian Oceans swordfish represent two distinct genetic stocks. Indo-Pacific differentiation was also significant but lower than that observed between Atlantic and Indian Oceans. However, microsatellite F-statistics failed to reveal structure even at the inter-oceanic scale, indicating that resolving power of our microsatellite loci was insufficient for detecting population subdivision. At the scale of the Indian Ocean, results obtained from both markers are consistent with swordfish belonging to a single unique panmictic population. Analyses partitioned by sampling area, season, or sex also failed to identify any clear structure within this ocean. Such large spatial and temporal homogeneity of genetic structure, observed for such a large highly mobile pelagic species, suggests as satisfactory to consider swordfish as a single panmictic population in the Indian Ocean. PMID:23717447

Single cell elemental analysis using nuclear microscopy

NASA Astrophysics Data System (ADS)

Ren, M. Q.; Thong, P. S. P.; Kara, U.; Watt, F.

1999-04-01

The use of Particle Induced X-ray Emission (PIXE), Rutherford Backscattering Spectrometry (RBS) and Scanning Transmission Ion Microscopy (STIM) to provide quantitative elemental analysis of single cells is an area which has high potential, particularly when the trace elements such as Ca, Fe, Zn and Cu can be monitored. We describe the methodology of sample preparation for two cell types, the procedures of cell imaging using STIM, and the quantitative elemental analysis of single cells using RBS and PIXE. Recent work on single cells at the Nuclear Microscopy Research Centre,National University of Singapore has centred around two research areas: (a) Apoptosis (programmed cell death), which has been recently implicated in a wide range of pathological conditions such as cancer, Parkinson's disease etc, and (b) Malaria (infection of red blood cells by the malaria parasite). Firstly we present results on the elemental analysis of human Chang liver cells (ATTCC CCL 13) where vanadium ions were used to trigger apoptosis, and demonstrate that nuclear microscopy has the capability of monitoring vanadium loading within individual cells. Secondly we present the results of elemental changes taking place in individual mouse red blood cells which have been infected with the malaria parasite and treated with the anti-malaria drug Qinghaosu (QHS).

Purposeful sampling for qualitative data collection and analysis in mixed method implementation research

PubMed Central

Palinkas, Lawrence A.; Horwitz, Sarah M.; Green, Carla A.; Wisdom, Jennifer P.; Duan, Naihua; Hoagwood, Kimberly

2013-01-01

Purposeful sampling is widely used in qualitative research for the identification and selection of information-rich cases related to the phenomenon of interest. Although there are several different purposeful sampling strategies, criterion sampling appears to be used most commonly in implementation research. However, combining sampling strategies may be more appropriate to the aims of implementation research and more consistent with recent developments in quantitative methods. This paper reviews the principles and practice of purposeful sampling in implementation research, summarizes types and categories of purposeful sampling strategies and provides a set of recommendations for use of single strategy or multistage strategy designs, particularly for state implementation research. PMID:24193818

Quantitative charge-tags for sterol and oxysterol analysis.

PubMed

Crick, Peter J; William Bentley, T; Abdel-Khalik, Jonas; Matthews, Ian; Clayton, Peter T; Morris, Andrew A; Bigger, Brian W; Zerbinati, Chiara; Tritapepe, Luigi; Iuliano, Luigi; Wang, Yuqin; Griffiths, William J

2015-02-01

Global sterol analysis is challenging owing to the extreme diversity of sterol natural products, the tendency of cholesterol to dominate in abundance over all other sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative charge-tags for the LC-MS analysis of sterols including oxysterols. Sterols/oxysterols in plasma were extracted in ethanol containing deuterated internal standards, separated by C18 solid-phase extraction, and derivatized with GP, with or without prior oxidation of 3β-hydroxy to 3-oxo groups. By use of different isotope-labeled GPs, it was possible to analyze in a single LC-MS analysis both sterols/oxysterols that naturally possess a 3-oxo group and those with a 3β-hydroxy group. Intra- and interassay CVs were <15%, and recoveries for representative oxysterols and cholestenoic acids were 85%-108%. By adopting a multiplex approach to isotope labeling, we analyzed up to 4 different samples in a single run. Using plasma samples, we could demonstrate the diagnosis of inborn errors of metabolism and also the export of oxysterols from brain via the jugular vein. This method allows the profiling of the widest range of sterols/oxysterols in a single analytical run and can be used to identify inborn errors of cholesterol synthesis and metabolism. © 2014 American Association for Clinical Chemistry.

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families.

PubMed

Bremner, P D; Blacklock, C J; Paganga, G; Mullen, W; Rice-Evans, C A; Crozier, A

2000-06-01

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.

Rapid Analysis of Trace Drugs and Metabolites Using a Thermal Desorption DART-MS Configuration.

PubMed

Sisco, Edward; Forbes, Thomas P; Staymates, Matthew E; Gillen, Greg

2016-01-01

The need to analyze trace narcotic samples rapidly for screening or confirmatory purposes is of increasing interest to the forensic, homeland security, and criminal justice sectors. This work presents a novel method for the detection and quantification of trace drugs and metabolites off of a swipe material using a thermal desorption direct analysis in real time mass spectrometry (TD-DART-MS) configuration. A variation on traditional DART, this configuration allows for desorption of the sample into a confined tube, completely independent of the DART source, allowing for more efficient and thermally precise analysis of material present on a swipe. Over thirty trace samples of narcotics, metabolites, and cutting agents deposited onto swipes were rapidly differentiated using this methodology. The non-optimized method led to sensitivities ranging from single nanograms to hundreds of picograms. Direct comparison to traditional DART with a subset of the samples highlighted an improvement in sensitivity by a factor of twenty to thirty and an increase in reproducibility sample to sample from approximately 45 % RSD to less than 15 % RSD. Rapid extraction-less quantification was also possible.

Comparison of mineral dust and droplet residuals measured with two single particle aerosol mass spectrometers

NASA Astrophysics Data System (ADS)

Wonaschütz, Anna; Ludwig, Wolfgang; Zawadowicz, Maria; Hiranuma, Naruki; Hitzenberger, Regina; Cziczo, Daniel; DeMott, Paul; Möhler, Ottmar

2017-04-01

Single Particle mass spectrometers are used to gain information on the chemical composition of individual aerosol particles, aerosol mixing state, and other valuable aerosol characteristics. During the Mass Spectrometry Intercomparison at the Fifth Ice Nucleation (FIN-01) Workshop, the new LAAPTOF single particle aerosol mass spectrometer (AeroMegt GmbH) was conducting simultaneous measurements together with the PALMS (Particle Analysis by Laser Mass Spectrometry) instrument. The aerosol particles were sampled from the AIDA chamber during ice cloud expansion experiments. Samples of mineral dust and ice droplet residuals were measured simultaneously. In this work, three expansion experiments are chosen for a comparison between the two mass spectrometers. A fuzzy clustering routine is used to group the spectra. Cluster centers describing the ensemble of particles are compared. First results show that while differences in the peak heights are likely due to the use of an amplifier in PALMS, cluster centers are comparable.

Charged particle induced delayed X-rays (DEX) for the analysis of intermediate and heavy elements

NASA Astrophysics Data System (ADS)

Pillay, A. E.; Erasmus, C. S.; Andeweg, A. H.; Sellschop, J. P. F.; Annegarn, H. J.; Dunn, J.

1988-12-01

The emission of K X-rays from proton-rich and metastable radionuclides, following proton activation of the stable isotopes of the elements of interest, has not been widely used as a means of analysis. The thrust of this paper proposes a nuclear technique using delayed X-rays for the analysis of low concentrations of intermediate and heavy elements. The method is similar to the delayed gamma-ray technique. Proton bombardment induces mainly (p, n) reactions whereas the delayed X-rays originate largely from e --capture and isomeric transition. Samples of rare earth and platinum group elements (PGE), in the form of compacted powders, were irradiated with an 11 MeV proton beam and delayed X-rays detected with a 100 mm 2 Ge detector. Single element spectra for a range of rare earths and PGEs are presented. Analytical conditions are demonstrated for Pd in the range 0.1-5%. Spectra from actual geological samples of a PGE ore, preconcentrated by fire-assay, and monazite are presented. All six platinum group elements are visible and interference-free in a single spectrum, a marked advance on other nuclear techniques for these elements, including PIXE and neutron activation analysis (NAA).

Uncertainty assessment method for the Cs-137 fallout inventory and penetration depth.

PubMed

Papadakos, G N; Karangelos, D J; Petropoulos, N P; Anagnostakis, M J; Hinis, E P; Simopoulos, S E

2017-05-01

Within the presented study, soil samples were collected in year 2007 at 20 different locations of the Greek terrain, both from the surface and also from depths down to 26 cm. Sampling locations were selected primarily from areas where high levels of 137 Cs deposition after the Chernobyl accident had already been identified by the Nuclear Engineering Laboratory of the National Technical University of Athens during and after the year of 1986. At one location of relatively higher deposition, soil core samples were collected following a 60 m by 60 m Cartesian grid with a 20 m node-to-node distance. Single or pair core samples were also collected from the remaining 19 locations. Sample measurements and analysis were used to estimate 137 Cs inventory and the corresponding depth migration, twenty years after the deposition on Greek terrain. Based on these data, the uncertainty components of the whole sampling-to-results procedure were investigated. A cause-and-effect assessment process was used to apply the law of error propagation and demonstrate that the dominating significant component of the combined uncertainty is that due to the spatial variability of the contemporary (2007) 137 Cs inventory. A secondary, yet also significant component was identified to be the activity measurement process itself. Other less-significant uncertainty parameters were sampling methods, the variation in the soil field density with depth and the preparation of samples for measurement. The sampling grid experiment allowed for the quantitative evaluation of the uncertainty due to spatial variability, also by the assistance of the semivariance analysis. Denser, optimized grid could return more accurate values for this component but with a significantly elevated laboratory cost, in terms of both, human and material resources. Using the hereby collected data and for the case of a single core soil sampling using a well-defined sampling methodology quality assurance, the uncertainty component due to spatial variability was evaluated to about 19% for the 137 Cs inventory and up to 34% for the 137 Cs penetration depth. Based on the presented results and also on related literature, it is argued that such high uncertainties should be anticipated for single core samplings conducted using similar methodology and employed as 137 Cs inventory and penetration depth estimators. Copyright © 2017 Elsevier Ltd. All rights reserved.

Research on width control of Metal Fused-coating Additive Manufacturing based on active control

NASA Astrophysics Data System (ADS)

Ren, Chuan qi; Wei, Zheng ying; Wang, Xin; Du, Jun; Zhang, Shan; Zhang, Zhitong; Bai, Hao

2017-12-01

Given the stability of the shape of the forming layer is one of the key problems that affect the final quality of the sample morphology, taking a study on the forming process and the control method of morphology make a significant difference to metal fused-coating additive manufacturing (MFCAM) in achieving the efficient and stable forming. To improve the quality and precision of the samples of single-layer single pass, a control method of morphology based on active control was established by this paper. The real-time acquisition of image was realized by CCD and the characteristics of morphology of the forming process were simultaneously extracted. Making analysis of the characteristics of the width during the process, the relationship between the relative difference of different frames and moving speed was given. A large number of experiments are used to verify the response speed and accuracy of the system. The results show that the active system can improve the morphology of the sample and the smoothness of the width of the single channel, and increase the uniformity of width by 55.16%.

Bi-PROF

PubMed Central

Gries, Jasmin; Schumacher, Dirk; Arand, Julia; Lutsik, Pavlo; Markelova, Maria Rivera; Fichtner, Iduna; Walter, Jörn; Sers, Christine; Tierling, Sascha

2013-01-01

The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 102-104 sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer. PMID:23803588

viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.

PubMed

Amir, El-ad David; Davis, Kara L; Tadmor, Michelle D; Simonds, Erin F; Levine, Jacob H; Bendall, Sean C; Shenfeld, Daniel K; Krishnaswamy, Smita; Nolan, Garry P; Pe'er, Dana

2013-06-01

New high-dimensional, single-cell technologies offer unprecedented resolution in the analysis of heterogeneous tissues. However, because these technologies can measure dozens of parameters simultaneously in individual cells, data interpretation can be challenging. Here we present viSNE, a tool that allows one to map high-dimensional cytometry data onto two dimensions, yet conserve the high-dimensional structure of the data. viSNE plots individual cells in a visual similar to a scatter plot, while using all pairwise distances in high dimension to determine each cell's location in the plot. We integrated mass cytometry with viSNE to map healthy and cancerous bone marrow samples. Healthy bone marrow automatically maps into a consistent shape, whereas leukemia samples map into malformed shapes that are distinct from healthy bone marrow and from each other. We also use viSNE and mass cytometry to compare leukemia diagnosis and relapse samples, and to identify a rare leukemia population reminiscent of minimal residual disease. viSNE can be applied to any multi-dimensional single-cell technology.

Frequency and characteristics of induced abortion among married and single women in São Paulo, Brazil.

PubMed

Souza e Silva, Rebeca de; Vieira, Elisabeth Meloni

2009-01-01

This article presents the results of a study in the city of São Paulo, Brazil, aimed at estimating the frequency of induced abortion among women 15 to 49 years of age. The objective was to characterize the occurrence of induced abortion by comparing the ideal number of children, age, and contraceptive use between married and single women. Based on random sampling, 1,749 interviews were held, including 764 married women, 658 single women, and 327 with other marital status. The analysis included: mean number of abortions per woman by analysis of variance and proportions of abortions and pregnancy, using the chi-square test. The mean abortion rate for married women (45 per thousand) did not differ statistically from that of single women. However, the pregnancy rate was much lower in single women, and when single women became pregnant they used abortion more frequently; while fewer than 2% of pregnancies in married women ended in induced abortions, among single women the abortion rate exceeded 18%. Therefore, the priority in the reproductive health field should be to invest in the supply and dissemination of appropriate contraceptive methods for women's early sexually active life.

Being Single as a Social Barrier to Access Reproductive Healthcare Services by Iranian Girls.

PubMed

Kohan, Shahnaz; Mohammadi, Fatemeh; Mostafavi, Firoozeh; Gholami, Ali

2016-08-17

Iranian single women are deprived of reproductive healthcare services, though the provision of such services to the public has increased. This study aimed to explore the experiences of Iranian single women on their access to reproductive health services. A qualitative design using a conventional content analysis method was used. Semi-structured interviews were held with 17 single women and nine health providers chosen using the purposive sampling method. Data analysis resulted in the development of three categories: 'family's attitudes and performance about single women's reproductive healthcare,' 'socio-cultural factors influencing reproductive healthcare,' and 'cultural factors influencing being a single woman.' Cultural and contextual factors affect being a single woman in every society. Therefore, healthcare providers need to identify such factors during the designing of strategies for improving the facilitation of access to reproductive healthcare services. © 2017 The Author(s); Published by Kerman University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Sensing of Streptococcus mutans by microscopic imaging ellipsometry

NASA Astrophysics Data System (ADS)

Khaleel, Mai Ibrahim; Chen, Yu-Da; Chien, Ching-Hang; Chang, Yia-Chung

2017-05-01

Microscopic imaging ellipsometry is an optical technique that uses an objective and sensing procedure to measure the ellipsometric parameters Ψ and Δ in the form of microscopic maps. This technique is well known for being noninvasive and label-free. Therefore, it can be used to detect and characterize biological species without any impact. Microscopic imaging ellipsometry was used to measure the optical response of dried Streptococcus mutans cells on a glass substrate. The ellipsometric Ψ and Δ maps were obtained with the Optrel Multiskop system for specular reflection in the visible range (λ=450 to 750 nm). The Ψ and Δ images at 500, 600, and 700 nm were analyzed using three different theoretical models with single-bounce, two-bounce, and multibounce light paths to obtain the optical constants and height distribution. The obtained images of the optical constants show different aspects when comparing the single-bounce analysis with the two-bounce or multibounce analysis in detecting S. mutans samples. Furthermore, the height distributions estimated by two-bounce and multibounce analyses of S. mutans samples were in agreement with the thickness values measured by AFM, which implies that the two-bounce and multibounce analyses can provide information complementary to that obtained by a single-bounce light path.

Chemical vapour deposition growth and Raman characterization of graphene layers and carbon nanotubes

NASA Astrophysics Data System (ADS)

Lai, Y.-C.; Rafailov, P. M.; Vlaikova, E.; Marinova, V.; Lin, S. H.; Yu, P.; Yu, S.-C.; Chi, G. C.; Dimitrov, D.; Sveshtarov, P.; Mehandjiev, V.; Gospodinov, M. M.

2016-02-01

Single-layer graphene films were grown by chemical vapour deposition (CVD) on Cu foil. The CVD process was complemented by plasma enhancement to grow also vertically aligned multiwalled carbon nanotubes using Ni nanoparticles as catalyst. The obtained samples were characterized by Raman spectroscopy analysis. Nature of defects in the samples and optimal growth conditions leading to achieve high quality of graphene and carbon nanotubes are discussed.

Qualitative and Quantitative Analysis of Proteome and Peptidome of Human Follicular Fluid Using Multiple Samples from Single Donor with LC-MS and SWATH Methodology.

PubMed

Lewandowska, Aleksandra E; Macur, Katarzyna; Czaplewska, Paulina; Liss, Joanna; Łukaszuk, Krzysztof; Ołdziej, Stanisław

2017-08-04

Human follicular fluid (hFF) is a natural environment of oocyte maturation, and some components of hFF could be used to judge oocyte capability for fertilization and further development. In our pilot small-scale study three samples from four donors (12 samples in total) were analyzed to determine which hFF proteins/peptides could be used to differentiate individual oocytes and which are patient-specific. Ultrafiltration was used to fractionate hFF to high-molecular-weight (HMW) proteome (>10 kDa) and low-molecular-weight (LMW) peptidome (<10 kDa) fractions. HMW and LMW compositions were analyzed using LC-MS in SWATH data acquisition and processing methodology. In total we were able to identify 158 proteins, from which 59 were never reported before as hFF components. 55 (45 not reported before) proteins were found by analyzing LMW fraction, 67 (14 not reported before) were found by analyzing HMW fraction, and 36 were identified in both fractions of hFF. We were able to perform quantitative analysis for 72 proteins from HMW fraction of hFF. We found that concentrations of 11 proteins varied substantially among hFF samples from single donors, and those proteins are promising targets to identify biomarkers useful in oocyte quality assessment.

A comprehensive quality control workflow for paired tumor-normal NGS experiments.

PubMed

Schroeder, Christopher M; Hilke, Franz J; Löffler, Markus W; Bitzer, Michael; Lenz, Florian; Sturm, Marc

2017-06-01

Quality control (QC) is an important part of all NGS data analysis stages. Many available tools calculate QC metrics from different analysis steps of single sample experiments (raw reads, mapped reads and variant lists). Multi-sample experiments, as sequencing of tumor-normal pairs, require additional QC metrics to ensure validity of results. These multi-sample QC metrics still lack standardization. We therefore suggest a new workflow for QC of DNA sequencing of tumor-normal pairs. With this workflow well-known single-sample QC metrics and additional metrics specific for tumor-normal pairs can be calculated. The segmentation into different tools offers a high flexibility and allows reuse for other purposes. All tools produce qcML, a generic XML format for QC of -omics experiments. qcML uses quality metrics defined in an ontology, which was adapted for NGS. All QC tools are implemented in C ++ and run both under Linux and Windows. Plotting requires python 2.7 and matplotlib. The software is available under the 'GNU General Public License version 2' as part of the ngs-bits project: https://github.com/imgag/ngs-bits. christopher.schroeder@med.uni-tuebingen.de. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Evaluation of Nd-Loaded SnO2:F Films Coated via Spray Pyrolysis

NASA Astrophysics Data System (ADS)

Turgut, G.

2018-05-01

Thin layers of single (F)- and double (F/Nd)-incorporated tin oxide have been coated on glass substrate via spray pyrolysis. The structural, morphological, electrical, and optical features of F-incorporated samples were evaluated depending on the Nd loading. X-ray diffraction analysis revealed that samples had tetragonal tin oxide structure with (211) and (200) preferential directions. The crystallite size and strain values varied from 37.98 nm and 1.21 × 10-3 to 52.12 nm and 1.88 × 10-3. Scanning electron microscopy analysis showed that the samples consisted of pyramidal, polyhedral, and needle-shaped granules. The lowest sheet resistance value of 1.22 Ω was found for 1.8 at.% Nd + 25 at.% F-coloaded SnO2. However, the widest optical bandgap of 4.01 eV was observed for the single 25 at.% F-loaded sample. The Urbach tail and figure of merit also changed in the ranges of 664 meV to 1296 meV and 6.4 × 10-2 Ω-1 to 2.3 × 10-3 Ω-1, respectively. The results presented herein indicate that the character of F-doped tin oxide films can be controlled by Nd loading and that these films could be useful for technological applications.

Simple transmission Raman measurements using a single multivariate model for analysis of pharmaceutical samples contained in capsules of different colors.

PubMed

Lee, Yeojin; Kim, Jaejin; Lee, Sanguk; Woo, Young-Ah; Chung, Hoeil

2012-01-30

Direct transmission Raman measurements for analysis of pharmaceuticals in capsules are advantageous since they can be used to determine active pharmaceutical ingredient (API) concentrations in a non-destructive manner and with much less fluorescence background interference from the capsules themselves compared to conventional back-scattering measurements. If a single calibration model such as developed from spectra simply collected in glass vials could be used to determine API concentrations of samples contained in capsules of different colors rather than constructing individual models for each capsule color, the utility of transmission measurements would be further enhanced. To evaluate the feasibility, transmission Raman spectra of binary mixtures of ambroxol and lactose were collected in a glass vial and a partial least squares (PLS) model for the determination of ambroxol concentration was developed. Then, the model was directly applied to determine ambroxol concentrations of samples contained in capsules of 4 different colors (blue, green, white and yellow). Although the prediction performance was slightly degraded when the samples were placed in blue or green capsules, due to the presence of weak fluorescence, accurate determination of ambroxol was generally achieved in all cases. The prediction accuracy was also investigated when the thickness of the capsule was varied. Copyright © 2011 Elsevier B.V. All rights reserved.

Evaluation of Nd-Loaded SnO2:F Films Coated via Spray Pyrolysis

NASA Astrophysics Data System (ADS)

Turgut, G.

2018-07-01

Thin layers of single (F)- and double (F/Nd)-incorporated tin oxide have been coated on glass substrate via spray pyrolysis. The structural, morphological, electrical, and optical features of F-incorporated samples were evaluated depending on the Nd loading. X-ray diffraction analysis revealed that samples had tetragonal tin oxide structure with (211) and (200) preferential directions. The crystallite size and strain values varied from 37.98 nm and 1.21 × 10-3 to 52.12 nm and 1.88 × 10-3. Scanning electron microscopy analysis showed that the samples consisted of pyramidal, polyhedral, and needle-shaped granules. The lowest sheet resistance value of 1.22 Ω was found for 1.8 at.% Nd + 25 at.% F-coloaded SnO2. However, the widest optical bandgap of 4.01 eV was observed for the single 25 at.% F-loaded sample. The Urbach tail and figure of merit also changed in the ranges of 664 meV to 1296 meV and 6.4 × 10-2 Ω-1 to 2.3 × 10-3 Ω-1, respectively. The results presented herein indicate that the character of F-doped tin oxide films can be controlled by Nd loading and that these films could be useful for technological applications.

Single shot white light interference microscopy with colour fringe analysis for quantitative phase imaging of biological cells

NASA Astrophysics Data System (ADS)

Srivastava, Vishal; Mehta, D. S.

2013-02-01

To quantitatively obtain the phase map of Onion and human red blood cell (RBC) from white light interferogram we used Hilbert transform color fringe analysis technique. The three Red, Blue and Green color components are decomposed from single white light interferogram and Refractive index profile for Red, Blue and Green colour were computed in a completely non-invasive manner for Onion and human RBC. The present technique might be useful for non-invasive determination of the refractive index variation within cells and tissues and morphological features of sample with ease of operation and low cost.

Confirmatory factor analysis applied to the Force Concept Inventory

NASA Astrophysics Data System (ADS)

Eaton, Philip; Willoughby, Shannon D.

2018-06-01

In 1995, Huffman and Heller used exploratory factor analysis to draw into question the factors of the Force Concept Inventory (FCI). Since then several papers have been published examining the factors of the FCI on larger sets of student responses and understandable factors were extracted as a result. However, none of these proposed factor models have been verified to not be unique to their original sample through the use of independent sets of data. This paper seeks to confirm the factor models proposed by Scott et al. in 2012, and Hestenes et al. in 1992, as well as another expert model proposed within this study through the use of confirmatory factor analysis (CFA) and a sample of 20 822 postinstruction student responses to the FCI. Upon application of CFA using the full sample, all three models were found to fit the data with acceptable global fit statistics. However, when CFA was performed using these models on smaller sample sizes the models proposed by Scott et al. and Eaton and Willoughby were found to be far more stable than the model proposed by Hestenes et al. The goodness of fit of these models to the data suggests that the FCI can be scored on factors that are not unique to a single class. These scores could then be used to comment on how instruction methods effect the performance of students along a single factor and more in-depth analyses of curriculum changes may be possible as a result.

Ion microprobe measurement of strontium isotopes in calcium carbonate with application to salmon otoliths

USGS Publications Warehouse

Weber, P.K.; Bacon, C.R.; Hutcheon, I.D.; Ingram, B.L.; Wooden, J.L.

2005-01-01

The ion microprobe has the capability to generate high resolution, high precision isotopic measurements, but analysis of the isotopic composition of strontium, as measured by the 87Sr/ 86Sr ratio, has been hindered by isobaric interferences. Here we report the first high precision measurements of 87Sr/ 86Sr by ion microprobe in calcium carbonate samples with moderate Sr concentrations. We use the high mass resolving power (7000 to 9000 M.R.P.) of the SHRIMP-RG ion microprobe in combination with its high transmission to reduce the number of interfering species while maintaining sufficiently high count rates for precise isotopic measurements. The isobaric interferences are characterized by peak modeling and repeated analyses of standards. We demonstrate that by sample-standard bracketing, 87Sr/86Sr ratios can be measured in inorganic and biogenic carbonates with Sr concentrations between 400 and 1500 ppm with ???2??? external precision (2??) for a single analysis, and subpermil external precision with repeated analyses. Explicit correction for isobaric interferences (peak-stripping) is found to be less accurate and precise than sample-standard bracketing. Spatial resolution is ???25 ??m laterally and 2 ??m deep for a single analysis, consuming on the order of 2 ng of material. The method is tested on otoliths from salmon to demonstrate its accuracy and utility. In these growth-banded aragonitic structures, one-week temporal resolution can be achieved. The analytical method should be applicable to other calcium carbonate samples with similar Sr concentrations. Copyright ?? 2005 Elsevier Ltd.

Determination of Baylisascaris schroederi Infection in Wild Giant Pandas by an Accurate and Sensitive PCR/CE-SSCP Method

PubMed Central

Zhang, Wenping; Yie, Shangmian; Yue, Bisong; Zhou, Jielong; An, Renxiong; Yang, Jiangdong; Chen, Wangli; Wang, Chengdong; Zhang, Liang; Shen, Fujun; Yang, Guangyou; Hou, Rong; Zhang, Zhihe

2012-01-01

It has been recognized that other than habitat loss, degradation and fragmentation, the infection of the roundworm Baylisascaris schroederi (B. schroederi) is one of the major causes of death in wild giant pandas. However, the prevalence and intensity of the parasite infection has been inconsistently reported through a method that uses sedimentation-floatation followed by a microscope examination. This method fails to accurately determine infection because there are many bamboo residues and/or few B. schroederi eggs in the examined fecal samples. In the present study, we adopted a method that uses PCR and capillary electrophoresis combined with a single-strand conformation polymorphism analysis (PCR/CE-SSCP) to detect B. schroederi infection in wild giant pandas at a nature reserve, and compared it to the traditional microscope approach. The PCR specifically amplified a single band of 279-bp from both fecal samples and positive controls, which was confirmed by sequence analysis to correspond to the mitochondrial COII gene of B. schroederi. Moreover, it was demonstrated that the amount of genomic DNA was linearly correlated with the peak area of the CE-SSCP analysis. Thus, our adopted method can reliably detect the infectious prevalence and intensity of B. schroederi in wild giant pandas. The prevalence of B. schroederi was found to be 54% in the 91 fecal samples examined, and 48% in the fecal samples of 31 identified individual giant pandas. Infectious intensities of the 91 fecal samples were detected to range from 2.8 to 959.2 units/gram, and from 4.8 to 959.2 units/gram in the fecal samples of the 31 identified giant pandas. For comparison, by using the traditional microscope method, the prevalence of B. schroederi was found to be only 33% in the 91 fecal samples, 32% in the fecal samples of the 31 identified giant pandas, and no reliable infectious intensity was observed. PMID:22911871

Determination of Baylisascaris schroederi infection in wild giant pandas by an accurate and sensitive PCR/CE-SSCP method.

PubMed

Zhang, Wenping; Yie, Shangmian; Yue, Bisong; Zhou, Jielong; An, Renxiong; Yang, Jiangdong; Chen, Wangli; Wang, Chengdong; Zhang, Liang; Shen, Fujun; Yang, Guangyou; Hou, Rong; Zhang, Zhihe

2012-01-01

It has been recognized that other than habitat loss, degradation and fragmentation, the infection of the roundworm Baylisascaris schroederi (B. schroederi) is one of the major causes of death in wild giant pandas. However, the prevalence and intensity of the parasite infection has been inconsistently reported through a method that uses sedimentation-floatation followed by a microscope examination. This method fails to accurately determine infection because there are many bamboo residues and/or few B. schroederi eggs in the examined fecal samples. In the present study, we adopted a method that uses PCR and capillary electrophoresis combined with a single-strand conformation polymorphism analysis (PCR/CE-SSCP) to detect B. schroederi infection in wild giant pandas at a nature reserve, and compared it to the traditional microscope approach. The PCR specifically amplified a single band of 279-bp from both fecal samples and positive controls, which was confirmed by sequence analysis to correspond to the mitochondrial COII gene of B. schroederi. Moreover, it was demonstrated that the amount of genomic DNA was linearly correlated with the peak area of the CE-SSCP analysis. Thus, our adopted method can reliably detect the infectious prevalence and intensity of B. schroederi in wild giant pandas. The prevalence of B. schroederi was found to be 54% in the 91 fecal samples examined, and 48% in the fecal samples of 31 identified individual giant pandas. Infectious intensities of the 91 fecal samples were detected to range from 2.8 to 959.2 units/gram, and from 4.8 to 959.2 units/gram in the fecal samples of the 31 identified giant pandas. For comparison, by using the traditional microscope method, the prevalence of B. schroederi was found to be only 33% in the 91 fecal samples, 32% in the fecal samples of the 31 identified giant pandas, and no reliable infectious intensity was observed.

Microfluidic Platform for Parallel Single Cell Analysis for Diagnostic Applications.

PubMed

Le Gac, Séverine

2017-01-01

Cell populations are heterogeneous: they can comprise different cell types or even cells at different stages of the cell cycle and/or of biological processes. Furthermore, molecular processes taking place in cells are stochastic in nature. Therefore, cellular analysis must be brought down to the single cell level to get useful insight into biological processes, and to access essential molecular information that would be lost when using a cell population analysis approach. Furthermore, to fully characterize a cell population, ideally, information both at the single cell level and on the whole cell population is required, which calls for analyzing each individual cell in a population in a parallel manner. This single cell level analysis approach is particularly important for diagnostic applications to unravel molecular perturbations at the onset of a disease, to identify biomarkers, and for personalized medicine, not only because of the heterogeneity of the cell sample, but also due to the availability of a reduced amount of cells, or even unique cells. This chapter presents a versatile platform meant for the parallel analysis of individual cells, with a particular focus on diagnostic applications and the analysis of cancer cells. We first describe one essential step of this parallel single cell analysis protocol, which is the trapping of individual cells in dedicated structures. Following this, we report different steps of a whole analytical process, including on-chip cell staining and imaging, cell membrane permeabilization and/or lysis using either chemical or physical means, and retrieval of the cell molecular content in dedicated channels for further analysis. This series of experiments illustrates the versatility of the herein-presented platform and its suitability for various analysis schemes and different analytical purposes.

Quantification of lithium at ppm level in geological samples using nuclear reaction analysis.

PubMed

De La Rosa, Nathaly; Kristiansson, Per; Nilsson, E J Charlotta; Ros, Linus; Pallon, Jan; Skogby, Henrik

2018-01-01

Proton-induced reaction (p,α) is one type of nuclear reaction analysis (NRA) suitable especially for light element quantification. In the case of lithium quantification presented in this work, accelerated protons with an energy about of 850 keV were used to induce the 7 Li(p,α) 4 He reaction in standard reference and geological samples such as tourmaline and other Li-minerals. It is shown that this technique for lithium quantification allowed for measurement of concentrations down below one ppm. The possibility to relate the lithium content with the boron content in a single analysis was also demonstrated using tourmaline samples, both in absolute concentration and in lateral distribution. In addition, Particle induced X-ray emission (PIXE) was utilized as a complementary IBA technique for simultaneous mapping of elements heavier than sodium.

Two-phase Neogene extension in the northwestern basin and range recorded in a single thermochronology sample

USGS Publications Warehouse

Colgan, J.P.; Shuster, D.L.; Reiners, P.W.

2008-01-01

We use a combination of apatite 4He/3He, (U-Th)/ He, and fission-track thermochronology to date slip on the Surprise Valley fault in northeastern California by analyzing a single sample from the Warner Range in the footwall of the fault. This sample, a granitic clast from a conglomerate, yielded a fission-track age of 11.6 ?? 2.8 Ma and a (U-Th)/He age of 3.02 ?? 0.52 Ma. Geologic relationships indicate that this sample was buried to a depth of ???3.3 km prior to exhumation during slip on the Surprise Valley fault. Fission-track age and length data indicate that the sample was fully reset (>120 ??C) prior to exhumation, which began sometime after 14 Ma. A single aliquot of nine apatite grains was step-heated for 4He/3He analysis; modeling of the resulting 4He distribution indicates that cooling from <80 ??C to ???20 ??C occurred between 3 and 1 Ma. Interconsistent time-temperature (t-T) solutions to the combined 4He/3He, (U-Th)/He, and fission-track data require two distinct periods of cooling, consistent with non-continuous slip on the Surprise Valley fault. Early cooling and fault slip took place between 14 and 8 Ma, followed by more recent fault slip ca. 3 Ma. This timing is consistent with both local geologic relationships and with the regional timing of faulting along the western margin of the Basin and Range Province. These data demonstrate the resolving power of combined fission-track, (U-Th)/He, and 4He/3He thermochronometric data to extract low-temperature t-T information from a single sample close to Earth's surface. ?? 2008 The Geological Society of America.

Fully Integrated Microfluidic Device for Direct Sample-to-Answer Genetic Analysis

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Grodzinski, Piotr

Integration of microfluidics technology with DNA microarrays enables building complete sample-to-answer systems that are useful in many applications such as clinic diagnostics. In this chapter, a fully integrated microfluidic device [1] that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and a DNA microarray sensor to perform DNA analysis of complex biological sample solutions is present. This device can perform on-chip sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis) of complex biological sample solutions (such as whole blood), polymerase chain reaction, DNA hybridization, and electrochemical detection. A few novel microfluidic techniques were developed and employed. A micromix-ing technique based on a cavitation microstreaming principle was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads. This technique was also employed to accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from ~mL whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers. | Office of Cancer Genomics

Cancer.gov

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers.

Development of size-selective sampling of Bacillus anthracis surrogate spores from simulated building air intake mixtures for analysis via laser-induced breakdown spectroscopy.

PubMed

Gibb-Snyder, Emily; Gullett, Brian; Ryan, Shawn; Oudejans, Lukas; Touati, Abderrahmane

2006-08-01

Size-selective sampling of Bacillus anthracis surrogate spores from realistic, common aerosol mixtures was developed for analysis by laser-induced breakdown spectroscopy (LIBS). A two-stage impactor was found to be the preferential sampling technique for LIBS analysis because it was able to concentrate the spores in the mixtures while decreasing the collection of potentially interfering aerosols. Three common spore/aerosol scenarios were evaluated, diesel truck exhaust (to simulate a truck running outside of a building air intake), urban outdoor aerosol (to simulate common building air), and finally a protein aerosol (to simulate either an agent mixture (ricin/anthrax) or a contaminated anthrax sample). Two statistical methods, linear correlation and principal component analysis, were assessed for differentiation of surrogate spore spectra from other common aerosols. Criteria for determining percentages of false positives and false negatives via correlation analysis were evaluated. A single laser shot analysis of approximately 4 percent of the spores in a mixture of 0.75 m(3) urban outdoor air doped with approximately 1.1 x 10(5) spores resulted in a 0.04 proportion of false negatives. For that same sample volume of urban air without spores, the proportion of false positives was 0.08.

Population connectivity of the plating coral Agaricia lamarcki from southwest Puerto Rico

NASA Astrophysics Data System (ADS)

Hammerman, Nicholas M.; Rivera-Vicens, Ramon E.; Galaska, Matthew P.; Weil, Ernesto; Appledoorn, Richard S.; Alfaro, Monica; Schizas, Nikolaos V.

2018-03-01

Identifying genetic connectivity and discrete population boundaries is an important objective for management of declining Caribbean reef-building corals. A double digest restriction-associated DNA sequencing protocol was utilized to generate 321 single nucleotide polymorphisms to estimate patterns of horizontal and vertical gene flow in the brooding Caribbean plate coral, Agaricia lamarcki. Individual colonies ( n = 59) were sampled from eight locations throughout southwestern Puerto Rico from six shallow ( 10-20 m) and two mesophotic habitats ( 30-40 m). Descriptive summary statistics (fixation index, F ST), analysis of molecular variance, and analysis through landscape and ecological associations and discriminant analysis of principal components estimated high population connectivity with subtle subpopulation structure among all sampling localities.

MASTR-MS: a web-based collaborative laboratory information management system (LIMS) for metabolomics.

PubMed

Hunter, Adam; Dayalan, Saravanan; De Souza, David; Power, Brad; Lorrimar, Rodney; Szabo, Tamas; Nguyen, Thu; O'Callaghan, Sean; Hack, Jeremy; Pyke, James; Nahid, Amsha; Barrero, Roberto; Roessner, Ute; Likic, Vladimir; Tull, Dedreia; Bacic, Antony; McConville, Malcolm; Bellgard, Matthew

2017-01-01

An increasing number of research laboratories and core analytical facilities around the world are developing high throughput metabolomic analytical and data processing pipelines that are capable of handling hundreds to thousands of individual samples per year, often over multiple projects, collaborations and sample types. At present, there are no Laboratory Information Management Systems (LIMS) that are specifically tailored for metabolomics laboratories that are capable of tracking samples and associated metadata from the beginning to the end of an experiment, including data processing and archiving, and which are also suitable for use in large institutional core facilities or multi-laboratory consortia as well as single laboratory environments. Here we present MASTR-MS, a downloadable and installable LIMS solution that can be deployed either within a single laboratory or used to link workflows across a multisite network. It comprises a Node Management System that can be used to link and manage projects across one or multiple collaborating laboratories; a User Management System which defines different user groups and privileges of users; a Quote Management System where client quotes are managed; a Project Management System in which metadata is stored and all aspects of project management, including experimental setup, sample tracking and instrument analysis, are defined, and a Data Management System that allows the automatic capture and storage of raw and processed data from the analytical instruments to the LIMS. MASTR-MS is a comprehensive LIMS solution specifically designed for metabolomics. It captures the entire lifecycle of a sample starting from project and experiment design to sample analysis, data capture and storage. It acts as an electronic notebook, facilitating project management within a single laboratory or a multi-node collaborative environment. This software is being developed in close consultation with members of the metabolomics research community. It is freely available under the GNU GPL v3 licence and can be accessed from, https://muccg.github.io/mastr-ms/.

Measuring discharge with ADCPs: Inferences from synthetic velocity profiles

USGS Publications Warehouse

Rehmann, C.R.; Mueller, D.S.; Oberg, K.A.

2009-01-01

Synthetic velocity profiles are used to determine guidelines for sampling discharge with acoustic Doppler current profilers (ADCPs). The analysis allows the effects of instrument characteristics, sampling parameters, and properties of the flow to be studied systematically. For mid-section measurements, the averaging time required for a single profile measurement always exceeded the 40 s usually recommended for velocity measurements, and it increased with increasing sample interval and increasing time scale of the large eddies. Similarly, simulations of transect measurements show that discharge error decreases as the number of large eddies sampled increases. The simulations allow sampling criteria that account for the physics of the flow to be developed. ?? 2009 ASCE.

Flexible nano- and microliter injections on a single liquid chromatography-mass spectrometry system: Minimizing sample preparation and maximizing linear dynamic range.

PubMed

Lubin, Arnaud; Sheng, Sheng; Cabooter, Deirdre; Augustijns, Patrick; Cuyckens, Filip

2017-11-17

Lack of knowledge on the expected concentration range or insufficient linear dynamic range of the analytical method applied are common challenges for the analytical scientist. Samples that are above the upper limit of quantification are typically diluted and reanalyzed. The analysis of undiluted highly concentrated samples can cause contamination of the system, while the dilution step is time consuming and as the case for any sample preparation step, also potentially leads to precipitation, adsorption or degradation of the analytes. Copyright © 2017 Elsevier B.V. All rights reserved.

Usefulness of the ARCHITECT Chagas® assay as a single test for the diagnosis of chronic Chagas disease.

PubMed

Pérez-Ayala, Ana; Fradejas, Isabel; Rebollo, Lourdes; Lora-Pablos, David; Lizasoain, Manuel; Herrero-Martínez, Juan María

2018-06-01

Imported Chagas disease (CD) is an emerging health problem in Europe due to immigration from endemic countries. Although WHO currently recommends two different serological methods to establish diagnosis, new tools like the ARCHITECT Chagas assay have potential for use as a single diagnostic test. Our objective was to determine an optimal signal-to-cut-off (S/CO) value for the ARCHITECT Chagas assay to diagnose CD with a single test. A retrospective study conducted at the 12 de Octubre University Hospital (Madrid, Spain). All patients with requests for Chagas screening between January 2014 and August 2017 were consecutively included. All samples were routinely tested with the ARCHITECT assay. Negative samples (S/CO < 0.8) required no further testing. Immunochromatographic testing (ICT) and/or indirect immunofluorescence (IFI) was used to confirm samples with S/CO ≥ 0.8. Receiver operator characteristic (ROC) curve analysis determined the ARCHITECT S/CO value that yielded 100% specificity and positive predictive value. SPSS software, version 22.0 was used for data analysis. A total of 4153 samples were analysed; 361 (8.69%) gave a reactive ARCHITECT Chagas result. 261/361 (72.3%) were women; median age was 38 years old (2-79). 92.8% were Bolivian. A total of 307 (85.0%) were confirmed as cases of Chagas; 52 (14.4%) were not infected; two (0.6%) were not evaluable. Seroprevalence was 7.39%. An S/CO ≥ 3.80 yielded 100% specificity (95% confidence interval [CI], 0.93-1.00) and 100% positive predictive value (95% CI, 0.99-1.00). Using S/CO ≥ 3.80, the ARCHITECT Chagas could be used as a single test for diagnosis of chronic CD in Bolivian immigrants. Patients with S/CO between 0.80 and 3.80 would require additional testing. © 2018 John Wiley & Sons Ltd.

Installation and Implementation of a Comprehensive Groundwater Monitoring Program for the Indian Wells Valley, California

DTIC Science & Technology

2010-04-01

isotopes. Laboratory analysis for general chemistry included Na, Ca, Mg, K, Fe, Cl, HCO3, CO3 , SO4, F, B, NO3, arsenic (As), hardness, alkalinity...used for interpretations within the project. Prior to this effort, a single -location repository for isotopic data related to IWV investigations...canyons of importance to this study (Indian Wells Canyon, Freeman Canyon, and the upgradient canyons of Cow Haven, Sage, and Horse). Single samples

A Review of Calibration Transfer Practices and Instrument Differences in Spectroscopy.

PubMed

Workman, Jerome J

2018-03-01

Calibration transfer for use with spectroscopic instruments, particularly for near-infrared, infrared, and Raman analysis, has been the subject of multiple articles, research papers, book chapters, and technical reviews. There has been a myriad of approaches published and claims made for resolving the problems associated with transferring calibrations; however, the capability of attaining identical results over time from two or more instruments using an identical calibration still eludes technologists. Calibration transfer, in a precise definition, refers to a series of analytical approaches or chemometric techniques used to attempt to apply a single spectral database, and the calibration model developed using that database, for two or more instruments, with statistically retained accuracy and precision. Ideally, one would develop a single calibration for any particular application, and move it indiscriminately across instruments and achieve identical analysis or prediction results. There are many technical aspects involved in such precision calibration transfer, related to the measuring instrument reproducibility and repeatability, the reference chemical values used for the calibration, the multivariate mathematics used for calibration, and sample presentation repeatability and reproducibility. Ideally, a multivariate model developed on a single instrument would provide a statistically identical analysis when used on other instruments following transfer. This paper reviews common calibration transfer techniques, mostly related to instrument differences, and the mathematics of the uncertainty between instruments when making spectroscopic measurements of identical samples. It does not specifically address calibration maintenance or reference laboratory differences.

High-Throughput Effect-Directed Analysis Using Downscaled in Vitro Reporter Gene Assays To Identify Endocrine Disruptors in Surface Water

PubMed Central

2018-01-01

Effect-directed analysis (EDA) is a commonly used approach for effect-based identification of endocrine disruptive chemicals in complex (environmental) mixtures. However, for routine toxicity assessment of, for example, water samples, current EDA approaches are considered time-consuming and laborious. We achieved faster EDA and identification by downscaling of sensitive cell-based hormone reporter gene assays and increasing fractionation resolution to allow testing of smaller fractions with reduced complexity. The high-resolution EDA approach is demonstrated by analysis of four environmental passive sampler extracts. Downscaling of the assays to a 384-well format allowed analysis of 64 fractions in triplicate (or 192 fractions without technical replicates) without affecting sensitivity compared to the standard 96-well format. Through a parallel exposure method, agonistic and antagonistic androgen and estrogen receptor activity could be measured in a single experiment following a single fractionation. From 16 selected candidate compounds, identified through nontargeted analysis, 13 could be confirmed chemically and 10 were found to be biologically active, of which the most potent nonsteroidal estrogens were identified as oxybenzone and piperine. The increased fractionation resolution and the higher throughput that downscaling provides allow for future application in routine high-resolution screening of large numbers of samples in order to accelerate identification of (emerging) endocrine disruptors. PMID:29547277

Sexual reproduction in Aspergillus tubingensis from section Nigri

USDA-ARS?s Scientific Manuscript database

A sclerotium-forming member of Aspergillus section Nigri was sampled from a population in a single field in North Carolina, USA, and identified as A. tubingensis based on genealogical concordance analysis. Aspergillus tubingensis was shown to be heterothallic, with individual strains containing ei...

Matrix-assisted laser desorption/ionization mass spectrometric imaging for the rapid segmental analysis of methamphetamine in a single hair using umbelliferone as a matrix.

PubMed

Wang, Hang; Wang, Ying

2017-07-04

Segmental hair analysis offers a longer period for retrospective drug detection than blood or urine. Hair is a keratinous fiber and is strongly hydrophobic. The embedding of drugs in hydrophobic hair at low concentrations makes it difficult for extraction and detection with matrix-assisted laser desorption/ionization (MALDI) coupled with mass spectrometric imaging (MSI). In this study, a single scalp hair was longitudinally cut with a cryostat section to a length of 4 mm and fixed onto a stainless steel MALDI plate. Umbelliferone was used as a new hydrophobic matrix to enrich and assist the ionization efficiency of methamphetamine in the hair sample. MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS profiling and imaging were performed for direct detection and mapping of methamphetamine on the longitudinal sections of the single hair sample in positive ion mode. Using MALDI-MSI, the distribution of methamphetamine was observed throughout five longitudinally sectioned hair samples from a drug abuser. The changes of methamphetamine were also semi-quantified by comparing the ratios of methamphetamine/internal standard (I.S). This method improves the detection sensitivity of target drugs embedded in a hair matrix for imaging with mass spectrometry. The method could provide a detection level of methamphetamine down to a nanogram per milligram incorporated into hair. The results were also compared with the conventional high performance liquid chromatography -tandem mass spectrometry (HPLC-MS/MS) method. Changes in the imaging results over time by the MSI method showed good semi-quantitative correlation to the results from the HPLC-MS/MS method. This study provides a powerful tool for drug abuse control and forensic medicine analysis in a narrow time frame, and a reduction in the sample amount required. Copyright © 2017 Elsevier B.V. All rights reserved.

iSBatch: a batch-processing platform for data analysis and exploration of live-cell single-molecule microscopy images and other hierarchical datasets.

PubMed

Caldas, Victor E A; Punter, Christiaan M; Ghodke, Harshad; Robinson, Andrew; van Oijen, Antoine M

2015-10-01

Recent technical advances have made it possible to visualize single molecules inside live cells. Microscopes with single-molecule sensitivity enable the imaging of low-abundance proteins, allowing for a quantitative characterization of molecular properties. Such data sets contain information on a wide spectrum of important molecular properties, with different aspects highlighted in different imaging strategies. The time-lapsed acquisition of images provides information on protein dynamics over long time scales, giving insight into expression dynamics and localization properties. Rapid burst imaging reveals properties of individual molecules in real-time, informing on their diffusion characteristics, binding dynamics and stoichiometries within complexes. This richness of information, however, adds significant complexity to analysis protocols. In general, large datasets of images must be collected and processed in order to produce statistically robust results and identify rare events. More importantly, as live-cell single-molecule measurements remain on the cutting edge of imaging, few protocols for analysis have been established and thus analysis strategies often need to be explored for each individual scenario. Existing analysis packages are geared towards either single-cell imaging data or in vitro single-molecule data and typically operate with highly specific algorithms developed for particular situations. Our tool, iSBatch, instead allows users to exploit the inherent flexibility of the popular open-source package ImageJ, providing a hierarchical framework in which existing plugins or custom macros may be executed over entire datasets or portions thereof. This strategy affords users freedom to explore new analysis protocols within large imaging datasets, while maintaining hierarchical relationships between experiments, samples, fields of view, cells, and individual molecules.

A novel method for single sample multi-axial nanoindentation of hydrated heterogeneous tissues based on testing great white shark jaws.

PubMed

Ferrara, Toni L; Boughton, Philip; Slavich, Eve; Wroe, Stephen

2013-01-01

Nanomechanical testing methods that are suitable for a range of hydrated tissues are crucial for understanding biological systems. Nanoindentation of tissues can provide valuable insights into biology, tissue engineering and biomimetic design. However, testing hydrated biological samples still remains a significant challenge. Shark jaw cartilage is an ideal substrate for developing a method to test hydrated tissues because it is a unique heterogeneous composite of both mineralized (hard) and non-mineralized (soft) layers and possesses a jaw geometry that is challenging to test mechanically. The aim of this study is to develop a novel method for obtaining multidirectional nanomechanical properties for both layers of jaw cartilage from a single sample, taken from the great white shark (Carcharodon carcharias). A method for obtaining multidirectional data from a single sample is necessary for examining tissue mechanics in this shark because it is a protected species and hence samples may be difficult to obtain. Results show that this method maintains hydration of samples that would otherwise rapidly dehydrate. Our study is the first analysis of nanomechanical properties of great white shark jaw cartilage. Variation in nanomechanical properties were detected in different orthogonal directions for both layers of jaw cartilage in this species. The data further suggest that the mineralized layer of shark jaw cartilage is less stiff than previously posited. Our method allows multidirectional nanomechanical properties to be obtained from a single, small, hydrated heterogeneous sample. Our technique is therefore suitable for use when specimens are rare, valuable or limited in quantity, such as samples obtained from endangered species or pathological tissues. We also outline a method for tip-to-optic calibration that facilitates nanoindentation of soft biological tissues. Our technique may help address the critical need for a nanomechanical testing method that is applicable to a variety of hydrated biological materials whether soft or hard.

Performance Enhancement of 3-Mercaptopropionic Acid-Capped CdSe Quantum-Dot Sensitized Solar Cells Incorporating Single-Walled Carbon Nanotubes.

PubMed

Yang, Jonghee; Park, Taehee; Lee, Jongtaek; Lee, Junyoung; Shin, Hokyeong; Yi, Whikun

2016-03-01

We fabricated a series of linker-assisted quantum-dot-sensitized solar cells based on the ex situ self-assembly of CdSe quantum dots (QDs) onto TiO2 electrode using sulfide/polysulfide (S(2-)/Sn(2-)) as an electrolyte and Au cathode. Our cell were combined with single-walled carbon nanotubes (SWNTs) by two techniques; One was mixing SWNTs with TiO2 electrode and the other was spraying SWNTs onto Au electrode. Absorption spectra were used to confirm the adsorption of QDs onto TiO2 electrode. Cell performance was measured on samples containing and not-containing SWNTs. Samples mixing SWNTs with TiO2 showed higher cell efficiency, on the while sample spraying SWNTs onto Au electrode showed lower efficiency compared with pristine sample (not-containing SWNTs). Electrochemical impedance spectroscopy analysis suggested that SWNTs can act as either barriers or excellent carrier transfers according their position and mixing method.

SciClone: inferring clonal architecture and tracking the spatial and temporal patterns of tumor evolution.

PubMed

Miller, Christopher A; White, Brian S; Dees, Nathan D; Griffith, Malachi; Welch, John S; Griffith, Obi L; Vij, Ravi; Tomasson, Michael H; Graubert, Timothy A; Walter, Matthew J; Ellis, Matthew J; Schierding, William; DiPersio, John F; Ley, Timothy J; Mardis, Elaine R; Wilson, Richard K; Ding, Li

2014-08-01

The sensitivity of massively-parallel sequencing has confirmed that most cancers are oligoclonal, with subpopulations of neoplastic cells harboring distinct mutations. A fine resolution view of this clonal architecture provides insight into tumor heterogeneity, evolution, and treatment response, all of which may have clinical implications. Single tumor analysis already contributes to understanding these phenomena. However, cryptic subclones are frequently revealed by additional patient samples (e.g., collected at relapse or following treatment), indicating that accurately characterizing a tumor requires analyzing multiple samples from the same patient. To address this need, we present SciClone, a computational method that identifies the number and genetic composition of subclones by analyzing the variant allele frequencies of somatic mutations. We use it to detect subclones in acute myeloid leukemia and breast cancer samples that, though present at disease onset, are not evident from a single primary tumor sample. By doing so, we can track tumor evolution and identify the spatial origins of cells resisting therapy.

SciClone: Inferring Clonal Architecture and Tracking the Spatial and Temporal Patterns of Tumor Evolution

PubMed Central

Dees, Nathan D.; Griffith, Malachi; Welch, John S.; Griffith, Obi L.; Vij, Ravi; Tomasson, Michael H.; Graubert, Timothy A.; Walter, Matthew J.; Ellis, Matthew J.; Schierding, William; DiPersio, John F.; Ley, Timothy J.; Mardis, Elaine R.; Wilson, Richard K.; Ding, Li

2014-01-01

The sensitivity of massively-parallel sequencing has confirmed that most cancers are oligoclonal, with subpopulations of neoplastic cells harboring distinct mutations. A fine resolution view of this clonal architecture provides insight into tumor heterogeneity, evolution, and treatment response, all of which may have clinical implications. Single tumor analysis already contributes to understanding these phenomena. However, cryptic subclones are frequently revealed by additional patient samples (e.g., collected at relapse or following treatment), indicating that accurately characterizing a tumor requires analyzing multiple samples from the same patient. To address this need, we present SciClone, a computational method that identifies the number and genetic composition of subclones by analyzing the variant allele frequencies of somatic mutations. We use it to detect subclones in acute myeloid leukemia and breast cancer samples that, though present at disease onset, are not evident from a single primary tumor sample. By doing so, we can track tumor evolution and identify the spatial origins of cells resisting therapy. PMID:25102416

Ultrasensitive Detection of Shigella Species in Blood and Stool.

PubMed

Luo, Jieling; Wang, Jiapeng; Mathew, Anup S; Yau, Siu-Tung

2016-02-16

A modified immunosensing system with voltage-controlled signal amplification was used to detect Shigella in stool and blood matrixes at the single-digit CFU level. Inactivated Shigella was spiked in these matrixes and detected directly. The detection was completed in 78 min. Detection limits of 21 CFU/mL and 18 CFU/mL were achieved in stool and blood, respectively, corresponding to 2-7 CFUs immobilized on the detecting electrode. The outcome of the detection of extremely low bacterium concentration, i.e., below 100 CFU/mL, blood samples show a random nature. An analysis of the detection probabilities indicates the correlation between the sample volume and the success of detection and suggests that sample volume is critical for ultrasensitive detection of bacteria. The calculated detection limit is qualitatively in agreement with the empirically determined detection limit. The demonstrated ultrasensitive detection of Shigella on the single-digit CFU level suggests the feasibility of the direct detection of the bacterium in the samples without performing a culture.

A comparison of four-sample slope-intercept and single-sample 51Cr-EDTA glomerular filtration rate measurements.

PubMed

Porter, Charlotte A; Bradley, Kevin M; McGowan, Daniel R

2018-05-01

The aim of this study was to verify, with a large dataset of 1394 Cr-EDTA glomerular filtration rate (GFR) studies, the equivalence of slope-intercept and single-sample GFR. Raw data from 1394 patient studies were used to calculate four-sample slope-intercept GFR in addition to four individual single-sample GFR values (blood samples taken at 90, 150, 210 and 270 min after injection). The percentage differences between the four-sample slope-intercept and each of the single-sample GFR values were calculated, to identify the optimum single-sample time point. Having identified the optimum time point, the percentage difference between the slope-intercept and optimal single-sample GFR was calculated across a range of GFR values to investigate whether there was a GFR value below which the two methodologies cannot be considered equivalent. It was found that the lowest percentage difference between slope-intercept and single-sample GFR was for the third blood sample, taken at 210 min after injection. The median percentage difference was 2.5% and only 6.9% of patient studies had a percentage difference greater than 10%. Above a GFR value of 30 ml/min/1.73 m, the median percentage difference between the slope-intercept and optimal single-sample GFR values was below 10%, and so it was concluded that, above this value, the two techniques are sufficiently equivalent. This study supports the recommendation of performing single-sample GFR measurements for GFRs greater than 30 ml/min/1.73 m.

DAPNe with micro-capillary separatory chemistry-coupled to MALDI-MS for the analysis of polar and non-polar lipid metabolism in one cell

NASA Astrophysics Data System (ADS)

Hamilton, Jason S.; Aguilar, Roberto; Petros, Robby A.; Verbeck, Guido F.

2017-05-01

The cellular metabolome is considered to be a representation of cellular phenotype and cellular response to changes to internal or external events. Methods to expand the coverage of the expansive physiochemical properties that makeup the metabolome currently utilize multi-step extractions and chromatographic separations prior to chemical detection, leading to lengthy analysis times. In this study, a single-step procedure for the extraction and separation of a sample using a micro-capillary as a separatory funnel to achieve analyte partitioning within an organic/aqueous immiscible solvent system is described. The separated analytes are then spotted for MALDI-MS imaging and distribution ratios are calculated. Initially, the method is applied to standard mixtures for proof of partitioning. The extraction of an individual cell is non-reproducible; therefore, a broad chemical analysis of metabolites is necessary and will be illustrated with the one-cell analysis of a single Snu-5 gastric cancer cell taken from a cellular suspension. The method presented here shows a broad partitioning dynamic range as a single-step method for lipid analysis demonstrating a decrease in ion suppression often present in MALDI analysis of lipids.

Quantitative determination of low-Z elements in single atmospheric particles on boron substrates by automated scanning electron microscopy-energy-dispersive X-ray spectrometry.

PubMed

Choël, Marie; Deboudt, Karine; Osán, János; Flament, Pascal; Van Grieken, René

2005-09-01

Atmospheric aerosols consist of a complex heterogeneous mixture of particles. Single-particle analysis techniques are known to provide unique information on the size-resolved chemical composition of aerosols. A scanning electron microscope (SEM) combined with a thin-window energy-dispersive X-ray (EDX) detector enables the morphological and elemental analysis of single particles down to 0.1 microm with a detection limit of 1-10 wt %, low-Z elements included. To obtain data statistically representative of the air masses sampled, a computer-controlled procedure can be implemented in order to run hundreds of single-particle analyses (typically 1000-2000) automatically in a relatively short period of time (generally 4-8 h, depending on the setup and on the particle loading). However, automated particle analysis by SEM-EDX raises two practical challenges: the accuracy of the particle recognition and the reliability of the quantitative analysis, especially for micrometer-sized particles with low atomic number contents. Since low-Z analysis is hampered by the use of traditional polycarbonate membranes, an alternate choice of substrate is a prerequisite. In this work, boron is being studied as a promising material for particle microanalysis. As EDX is generally said to probe a volume of approximately 1 microm3, geometry effects arise from the finite size of microparticles. These particle geometry effects must be corrected by means of a robust concentration calculation procedure. Conventional quantitative methods developed for bulk samples generate elemental concentrations considerably in error when applied to microparticles. A new methodology for particle microanalysis, combining the use of boron as the substrate material and a reverse Monte Carlo quantitative program, was tested on standard particles ranging from 0.25 to 10 microm. We demonstrate that the quantitative determination of low-Z elements in microparticles is achievable and that highly accurate results can be obtained using the automatic data processing described here compared to conventional methods.

Characterisation of Fine Ash Fractions from the AD 1314 Kaharoa Eruption

NASA Astrophysics Data System (ADS)

Weaver, S. J.; Rust, A.; Carey, R. J.; Houghton, B. F.

2012-12-01

The AD 1314±12 yr Kaharoa eruption of Tarawera volcano, New Zealand, produced deposits exhibiting both plinian and subplinian characteristics (Nairn et al., 2001; 2004, Leonard et al., 2002, Hogg et al., 2003). Their widespread dispersal yielded volumes, column heights, and mass discharge rates of plinian magnitude and intensity (Sahetapy-Engel, 2002); however, vertical shifts in grain size suggest waxing and waning within single phases and time-breaks on the order of hours between phases. These grain size shifts were quantified using sieve, laser diffraction, and image analysis of the fine ash fractions (<1 mm in diameter) of some of the most explosive phases of the eruption. These analyses served two purposes: 1) to characterise the change in eruption intensity over time, and 2) to compare the three methods of grain size analysis. Additional analyses of the proportions of components and particle shape were also conducted to aid in the interpretation of the eruption and transport dynamics. 110 samples from a single location about 6 km from source were sieved at half phi intervals between -4φ to 4φ (16 mm - 63 μm). A single sample was then chosen to test the range of grain sizes to run through the Mastersizer 2000. Three aliquots were tested; the first consisted of each sieve size fraction ranging between 0φ (1000 μm) and <4φ (<63 μm, i.e. the pan). For example, 0, 0.5, 1, …, 4φ, and the pan were ran through the Mastersizer and then their results, weighted according to their sieve weight percents, were summed together to produce a total distribution. The second aliquot included 3 samples ranging between 0-2φ (1000-250 μm), 2.5-4φ (249-63 μm), and the pan. A single sample consisting of the total range of grain sizes between 0φ and the pan was used for the final aliquot. Their results were compared and it was determined that the single sample consisting of the broadest range of grain sizes yielded an accurate grain size distribution. This data was then compared with the sieve weight percent data, and revealed that there is a significant difference in size characterisation between sieving and the Mastersizer for size fractions between 0-3φ (1000-125 μm). This is due predominantly to the differing methods that sieving and the Mastersizer use to characterise a single particle, to inhomogeneity in grain density in each grain-size fraction, and to grain-shape irregularities. This led the Mastersizer to allocate grains from a certain sieve size fraction into coarser size fractions. Therefore, only the Mastersizer data from 3.5φ and below were combined with the coarser sieve data to yield total grain size distributions. This high-resolution analysis of the grain size data enabled subtle trends in grain size to be identified and related to short timescale eruptive processes.

High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples.

PubMed

Payne, Matthew S; Tabone, Tania; Kemp, Matthew W; Keelan, Jeffrey A; Spiller, O Brad; Newnham, John P

2014-02-01

Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

Identification of active fluorescence stained bacteria by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Krause, Mario; Beyer, Beatrice; Pietsch, Christian; Radt, Benno; Harz, Michaela; Rösch, Petra; Popp, Jürgen

2008-04-01

Microorganisms can be found everywhere e.g. in food both as useful ingredients or harmful contaminations causing food spoilage. Therefore, a fast and easy to handle analysis method is needed to detect bacteria in different kinds of samples like meat, juice or air to decide if the sample is contaminated by harmful microorganisms. Conventional identification methods in microbiology require always cultivation and therefore are time consuming. In this contribution we present an analysis approach to identify fluorescence stained bacteria on strain level by means of Raman spectroscopy. The stained bacteria are highlighted and can be localized easier against a complex sample environment e.g. in food. The use of Raman spectroscopy in combination with chemometrical methods allows the identification of single bacteria within minutes.

Analysis of suspended solids by single-particle scattering. [for Lake Superior pollution monitoring

NASA Technical Reports Server (NTRS)

Diehl, S. R.; Smith, D. T.; Sydor, M.

1979-01-01

Light scattering by individual particulates is used in a multiple-detector system to categorize the composition of suspended solids in terms of broad particulate categories. The scattering signatures of red clay and taconite tailings, the two primary particulate contaminants in western Lake Superior, along with two types of asbestiform fibers, amphibole and chrysolite, were studied in detail. A method was developed to predict the concentration of asbestiform fibers in filtration plant samples for which electron microscope analysis was done concurrently. Fiber levels as low as 50,000 fibers/liter were optically detectable. The method has application in optical categorization of samples for remote sensing purposes and offers a fast, inexpensive means for analyzing water samples from filtration plants for specific particulate contaminants.

Single-Molecule Counting of Point Mutations by Transient DNA Binding

NASA Astrophysics Data System (ADS)

Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

2017-03-01

High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing.

PubMed

Xu, Chun-Xiu; Yin, Xue-Feng

2011-02-04

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance. Copyright © 2010 Elsevier B.V. All rights reserved.

Routine conventional karyotyping of lymphoma staging bone marrow samples does not contribute clinically relevant information.

PubMed

Nardi, Valentina; Pulluqi, Olja; Abramson, Jeremy S; Dal Cin, Paola; Hasserjian, Robert P

2015-06-01

Bone marrow (BM) evaluation is an important part of lymphoma staging, which guides patient management. Although positive staging marrow is defined as morphologically identifiable disease, such samples often also include flow cytometric analysis and conventional karyotyping. Cytogenetic analysis is a labor-intensive and costly procedure and its utility in this setting is uncertain. We retrospectively reviewed pathological reports of 526 staging marrow specimens in which conventional karyotyping had been performed. All samples originated from a single institution from patients with previously untreated Hodgkin and non-Hodgkin lymphomas presenting in an extramedullary site. Cytogenetic analysis revealed clonal abnormalities in only eight marrow samples (1.5%), all of which were positive for lymphoma by morphologic evaluation. Flow cytometry showed a small clonal lymphoid population in three of the 443 morphologically negative marrow samples (0.7%). Conventional karyotyping is rarely positive in lymphoma staging marrow samples and, in our cohort, the BM karyotype did not contribute clinically relevant information in the vast majority of cases. Our findings suggest that karyotyping should not be performed routinely on BM samples taken to stage previously diagnosed extramedullary lymphomas unless there is pathological evidence of BM involvement by lymphoma. © 2015 Wiley Periodicals, Inc.

A combined method for DNA analysis and radiocarbon dating from a single sample.

PubMed

Korlević, Petra; Talamo, Sahra; Meyer, Matthias

2018-03-07

Current protocols for ancient DNA and radiocarbon analysis of ancient bones and teeth call for multiple destructive samplings of a given specimen, thereby increasing the extent of undesirable damage to precious archaeological material. Here we present a method that makes it possible to obtain both ancient DNA sequences and radiocarbon dates from the same sample material. This is achieved by releasing DNA from the bone matrix through incubation with either EDTA or phosphate buffer prior to complete demineralization and collagen extraction utilizing the acid-base-acid-gelatinization and ultrafiltration procedure established in most radiocarbon dating laboratories. Using a set of 12 bones of different ages and preservation conditions we demonstrate that on average 89% of the DNA can be released from sample powder with minimal, or 38% without any, detectable collagen loss. We also detect no skews in radiocarbon dates compared to untreated samples. Given the different material demands for radiocarbon dating (500 mg of bone/dentine) and DNA analysis (10-100 mg), combined DNA and collagen extraction not only streamlines the sampling process but also drastically increases the amount of DNA that can be recovered from limited sample material.

Microfluidic single-cell whole-transcriptome sequencing.

PubMed

Streets, Aaron M; Zhang, Xiannian; Cao, Chen; Pang, Yuhong; Wu, Xinglong; Xiong, Liang; Yang, Lu; Fu, Yusi; Zhao, Liang; Tang, Fuchou; Huang, Yanyi

2014-05-13

Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.

Design and calibration of a vacuum compatible scanning tunneling microscope

NASA Technical Reports Server (NTRS)

Abel, Phillip B.

1990-01-01

A vacuum compatible scanning tunneling microscope was designed and built, capable of imaging solid surfaces with atomic resolution. The single piezoelectric tube design is compact, and makes use of sample mounting stubs standard to a commercially available surface analysis system. Image collection and display is computer controlled, allowing storage of images for further analysis. Calibration results from atomic scale images are presented.

Niche and neutral processes both shape community structure in parallelized, aerobic, single carbon-source enrichments

DOE Data Explorer

Flynn, Theodore M.; Koval, Jason C.; Greenwald, Stephanie M.; Owens, Sarah M.; Kemner, Kenneth M.; Antonopoulos, Dionysios A.

2017-01-01

We present DNA sequence data in FASTA-formatted files from aerobic environmental microcosms inoculated with a sole carbon source. DNA sequences are of 16S rRNA genes present in DNA extracted from each microcosm along with the environmental samples (soil, water) used to inoculate them. These samples were sequenced using the Illumina MiSeq platform at the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory. This data is compatible with standard microbiome analysis pipelines (e.g., QIIME, mothur, etc.).

Demonstration of Multi- and Single-Reader Sample Size Program for Diagnostic Studies software.

PubMed

Hillis, Stephen L; Schartz, Kevin M

2015-02-01

The recently released software Multi- and Single-Reader Sample Size Sample Size Program for Diagnostic Studies , written by Kevin Schartz and Stephen Hillis, performs sample size computations for diagnostic reader-performance studies. The program computes the sample size needed to detect a specified difference in a reader performance measure between two modalities, when using the analysis methods initially proposed by Dorfman, Berbaum, and Metz (DBM) and Obuchowski and Rockette (OR), and later unified and improved by Hillis and colleagues. A commonly used reader performance measure is the area under the receiver-operating-characteristic curve. The program can be used with typical common reader-performance measures which can be estimated parametrically or nonparametrically. The program has an easy-to-use step-by-step intuitive interface that walks the user through the entry of the needed information. Features of the software include the following: (1) choice of several study designs; (2) choice of inputs obtained from either OR or DBM analyses; (3) choice of three different inference situations: both readers and cases random, readers fixed and cases random, and readers random and cases fixed; (4) choice of two types of hypotheses: equivalence or noninferiority; (6) choice of two output formats: power for specified case and reader sample sizes, or a listing of case-reader combinations that provide a specified power; (7) choice of single or multi-reader analyses; and (8) functionality in Windows, Mac OS, and Linux.

Impact of Tropopause Structures on Deep Convective Transport Observed during MACPEX

NASA Astrophysics Data System (ADS)

Mullendore, G. L.; Bigelbach, B. C.; Christensen, L. E.; Maddox, E.; Pinkney, K.; Wagner, S.

2016-12-01

Deep convection is the most efficient method of transporting boundary layer mass to the upper troposphere and stratosphere (UTLS). The Mid-latitude Airborne Cirrus Properties Experiment (MACPEX) was conducted during April of 2011 over the central U.S. With a focus on cirrus clouds, the campaign flights often sampled large cirrus anvils downstream from deep convection and included an extensive observational suite of chemical measurements on a high altitude aircraft. As double-tropopause structures are a common feature in the central U.S. during the springtime, the MACPEX campaign provides a good opportunity to gather cases of deep convective transport in the context of both single and double tropopause structures. Sampling of chemical plumes well downstream from convection allows for sampling in relatively quiescent conditions and analysis of irreversible transport. The analysis presented includes multiple methods to assess air mass source and possible convective processing, including back trajectories and ratios of chemical concentrations. Although missions were flown downstream of deep convection, recent processing by convection does not seem likely in all cases that high altitude carbon monoxide plumes were observed. Additionally, the impact of single and double tropopause structures on deep convective transport is shown to be strongly dependent on high stability layers.

A hybrid sensing approach for pure and adulterated honey classification.

PubMed

Subari, Norazian; Mohamad Saleh, Junita; Md Shakaff, Ali Yeon; Zakaria, Ammar

2012-10-17

This paper presents a comparison between data from single modality and fusion methods to classify Tualang honey as pure or adulterated using Linear Discriminant Analysis (LDA) and Principal Component Analysis (PCA) statistical classification approaches. Ten different brands of certified pure Tualang honey were obtained throughout peninsular Malaysia and Sumatera, Indonesia. Various concentrations of two types of sugar solution (beet and cane sugar) were used in this investigation to create honey samples of 20%, 40%, 60% and 80% adulteration concentrations. Honey data extracted from an electronic nose (e-nose) and Fourier Transform Infrared Spectroscopy (FTIR) were gathered, analyzed and compared based on fusion methods. Visual observation of classification plots revealed that the PCA approach able to distinct pure and adulterated honey samples better than the LDA technique. Overall, the validated classification results based on FTIR data (88.0%) gave higher classification accuracy than e-nose data (76.5%) using the LDA technique. Honey classification based on normalized low-level and intermediate-level FTIR and e-nose fusion data scored classification accuracies of 92.2% and 88.7%, respectively using the Stepwise LDA method. The results suggested that pure and adulterated honey samples were better classified using FTIR and e-nose fusion data than single modality data.

Comprehensive Analysis of Immunological Synapse Phenotypes Using Supported Lipid Bilayers.

PubMed

Valvo, Salvatore; Mayya, Viveka; Seraia, Elena; Afrose, Jehan; Novak-Kotzer, Hila; Ebner, Daniel; Dustin, Michael L

2017-01-01

Supported lipid bilayers (SLB) formed on glass substrates have been a useful tool for study of immune cell signaling since the early 1980s. The mobility of lipid-anchored proteins in the system, first described for antibodies binding to synthetic phospholipid head groups, allows for the measurement of two-dimensional binding reactions and signaling processes in a single imaging plane over time or for fixed samples. The fragility of SLB and the challenges of building and validating individual substrates limit most experimenters to ~10 samples per day, perhaps increasing this few-fold when examining fixed samples. Successful experiments might then require further days to fully analyze. We present methods for automation of many steps in SLB formation, imaging in 96-well glass bottom plates, and analysis that enables >100-fold increase in throughput for fixed samples and wide-field fluorescence. This increased throughput will allow better coverage of relevant parameters and more comprehensive analysis of aspects of the immunological synapse that are well reconstituted by SLB.

Statistically optimal analysis of state-discretized trajectory data from multiple thermodynamic states

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

Wu, Hao; Mey, Antonia S. J. S.; Noé, Frank

2014-12-07

We propose a discrete transition-based reweighting analysis method (dTRAM) for analyzing configuration-space-discretized simulation trajectories produced at different thermodynamic states (temperatures, Hamiltonians, etc.) dTRAM provides maximum-likelihood estimates of stationary quantities (probabilities, free energies, expectation values) at any thermodynamic state. In contrast to the weighted histogram analysis method (WHAM), dTRAM does not require data to be sampled from global equilibrium, and can thus produce superior estimates for enhanced sampling data such as parallel/simulated tempering, replica exchange, umbrella sampling, or metadynamics. In addition, dTRAM provides optimal estimates of Markov state models (MSMs) from the discretized state-space trajectories at all thermodynamic states. Under suitablemore » conditions, these MSMs can be used to calculate kinetic quantities (e.g., rates, timescales). In the limit of a single thermodynamic state, dTRAM estimates a maximum likelihood reversible MSM, while in the limit of uncorrelated sampling data, dTRAM is identical to WHAM. dTRAM is thus a generalization to both estimators.« less

Synthesis, crystal growth and characterization of a phase matchable nonlinear optical single crystal: p-chloro dibenzylideneacetone

NASA Astrophysics Data System (ADS)

Ravindra, H. J.; John Kiran, A.; Nooji, Satheesha Rai; Dharmaprakash, S. M.; Chandrasekharan, K.; Kalluraya, Balakrishna; Rotermund, Fabian

2008-05-01

Good quality single crystals of p-chloro dibenzylideneacetone (CDBA) of size 13 mm×8 mm×2 mm were grown by slow evaporation solution growth technique. The grown crystals were confirmed by elemental analysis, Fourier transform infrared (FTIR) analysis and single crystal X-ray diffraction techniques. From the thermo gravimetric/differential thermal (TG/DT) analysis, the CDBA was found to be thermally stable up to 250 °C. The mechanical stability of the crystal is comparable with that of the other reported chalcones. The lower optical cut-off wavelength for this crystal was observed at 440 nm. The laser damage threshold of the crystal was 0.6 GW/cm 2 at 532 nm. The second harmonic generation conversion efficiency of the powder sample of CDBA was found to be 4.5 times greater than that of urea. We also demonstrate the existence of the phase matching property in this crystal using Kurtz powder technique.

Application of biodynamic imaging for personalized chemotherapy in canine lymphoma

NASA Astrophysics Data System (ADS)

Custead, Michelle R.

Biodynamic imaging (BDI) is a novel phenotypic cancer profiling technology which characterizes changes in cellular and subcellular motion in living tumor tissue samples following in vitro or ex vivo treatment with chemotherapeutics. The ability of BDI to predict clinical response to single-agent doxorubicin chemotherapy was tested in ten dogs with naturally-occurring non-Hodgkin's lymphomas (NHL). Pre-treatment tumor biopsy samples were obtained from all dogs and treated with doxorubicin (10 muM) ex vivo. BDI captured cellular and subcellular motility measures on all biopsy samples at baseline and at regular intervals for 9 hours following drug application. All dogs subsequently received treatment with a standard single-agent doxorubicin protocol. Objective response (OR) to doxorubicin and progression-free survival time (PFST) following chemotherapy were recorded for all dogs. The dynamic biomarkers measured by BDI were entered into a multivariate logistic model to determine the extent to which BDI predicted OR and PFST following doxorubicin therapy. The model showed that the sensitivity, specificity, and accuracy of BDI for predicting treatment outcome were 95%, 91%, and 93%, respectively. To account for possible over-fitting of data to the predictive model, cross-validation with a one-left-out analysis was performed, and the adjusted sensitivity, specificity, and accuracy following this analysis were 93%, 87%, and 91%, respectively. These findings suggest that BDI can predict, with high accuracy, treatment outcome following single-agent doxorubicin chemotherapy in a relevant spontaneous canine cancer model, and is a promising novel technology for advancing personalized cancer medicine.

Not simply more of the same: distinguishing between patient heterogeneity and parameter uncertainty.

PubMed

Vemer, Pepijn; Goossens, Lucas M A; Rutten-van Mölken, Maureen P M H

2014-11-01

In cost-effectiveness (CE) Markov models, heterogeneity in the patient population is not automatically taken into account. We aimed to compare methods of dealing with heterogeneity on estimates of CE, using a case study in chronic obstructive pulmonary disease (COPD). We first present a probabilistic sensitivity analysis (PSA) in which we sampled only from distributions representing parameter uncertainty. This ignores any heterogeneity. Next, we explored heterogeneity by presenting results for subgroups, using a method that samples parameter uncertainty simultaneously with heterogeneity in a single-loop PSA. Finally, we distinguished parameter uncertainty from heterogeneity in a double-loop PSA by performing a nested simulation within each PSA iteration. Point estimates and uncertainty differed substantially between methods. The incremental CE ratio (ICER) ranged from € 4900 to € 13,800. The single-loop PSA led to a substantially different shape of the CE plane and an overestimation of the uncertainty compared with the other 3 methods. The CE plane for the double-loop PSA showed substantially less uncertainty and a stronger negative correlation between the difference in costs and the difference in effects compared with the other methods. This came at the cost of higher calculation times. Not accounting for heterogeneity, subgroup analysis and the double-loop PSA can be viable options, depending on the decision makers' information needs. The single-loop PSA should not be used in CE research. It disregards the fundamental differences between heterogeneity and sampling uncertainty and overestimates uncertainty as a result. © The Author(s) 2014.

Effectiveness of probiotic, chlorhexidine and fluoride mouthwash against Streptococcus mutans – Randomized, single-blind, in vivo study

PubMed Central

Jothika, Mohan; Vanajassun, P. Pranav; Someshwar, Battu

2015-01-01

Aim: To determine the short-term efficiency of probiotic, chlorhexidine, and fluoride mouthwashes on plaque Streptococcus mutans level at four periodic intervals. Materials and Methods: This was a single-blind, randomized control study in which each subject was tested with only one mouthwash regimen. Fifty-two healthy qualified adult patients were selected randomly for the study and were divided into the following groups: group 1- 10 ml of distilled water, group 2- 10 ml of 0.2% chlorhexidine mouthwash, group 3- 10 ml of 500 ppm F/400 ml sodium fluoride mouthwash, and group 4- 10 ml of probiotic mouthwash. Plaque samples were collected from the buccal surface of premolars and molars in the maxillary quadrant. Sampling procedure was carried out by a single examiner after 7 days, 14 days, and 30 days, respectively, after the use of the mouthwash. All the samples were subjected to microbiological analysis and statistically analyzed with one-way analysis of variance (ANOVA) and post-hoc test. Results: One-way ANOVA comparison among groups 2, 3, and 4 showed no statistical significance, whereas group 1 showed statistically significant difference when compared with groups 2, 3, and 4 at 7th, 14th, and 30th day. Conclusion: Chlorhexidine, sodium fluoride, and probiotic mouthwashes reduce plaque S. mutans levels. Probiotic mouthwash is effective and equivalent to chlorhexidine and sodium fluoride mouthwashes. Thus, probiotic mouthwash can also be considered as an effective oral hygiene regimen. PMID:25984467

Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities

Treesearch

Fei Cheng; Lin Hou; Keith Woeste; Zhengchun Shang; Xiaobang Peng; Peng Zhao; Shuoxin Zhang

2016-01-01

Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high...

Beam tracking approach for single–shot retrieval of absorption, refraction, and dark-field signals with laboratory  x-ray sources

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

Vittoria, Fabio A., E-mail: fabio.vittoria.12@ucl.ac.uk; Diemoz, Paul C.; Olivo, Alessandro

We present the translation of the beam tracking approach for x-ray phase-contrast and dark-field imaging, recently demonstrated using synchrotron radiation, to a laboratory setup. A single absorbing mask is used before the sample, and a local Gaussian interpolation of the beam at the detector is used to extract absorption, refraction, and dark–field signals from a single exposure of the sample. Multiple exposures can be acquired when high resolution is needed, as shown here. A theoretical analysis of the effect of polychromaticity on the retrieved signals, and of the artifacts this might cause when existing retrieval methods are used, is alsomore » discussed.« less

Combustion synthesis and structural analysis of nanocrystalline nickel ferrite at low temperature regime

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

Shanmugavel, T., E-mail: gokulrajs@hotmail.com, E-mail: shanmugavelnano@gmail.com; Raj, S. Gokul, E-mail: gokulrajs@hotmail.com, E-mail: shanmugavelnano@gmail.com; Rajarajan, G.

2015-06-24

Combustion synthesis of single phase Nickel ferrite was successfully achieved at low temperature regime. The obtained powders were calcinated to increase the crystallinity and their characterization change due to calcinations is investigated in detail. Citric acid used as a chelating agent for the synthesis of nickel ferrite. Pure single phase nickel ferrites were found at this low temperature. The average crystalline sizes were measured by using powder XRD measurements. Surface morphology was investigated through Transmission Electron Microscope (TEM). Particle size calculated in XRD is compared with TEM results. Magnetic behaviour of the samples is analyzed by using Vibrating Sample Magnetometermore » (VSM). Saturation magnetization, coercivity and retentivity are measured and their results are discussed in detail.« less

N-of-1-pathways MixEnrich: advancing precision medicine via single-subject analysis in discovering dynamic changes of transcriptomes.

PubMed

Li, Qike; Schissler, A Grant; Gardeux, Vincent; Achour, Ikbel; Kenost, Colleen; Berghout, Joanne; Li, Haiquan; Zhang, Hao Helen; Lussier, Yves A

2017-05-24

Transcriptome analytic tools are commonly used across patient cohorts to develop drugs and predict clinical outcomes. However, as precision medicine pursues more accurate and individualized treatment decisions, these methods are not designed to address single-patient transcriptome analyses. We previously developed and validated the N-of-1-pathways framework using two methods, Wilcoxon and Mahalanobis Distance (MD), for personal transcriptome analysis derived from a pair of samples of a single patient. Although, both methods uncover concordantly dysregulated pathways, they are not designed to detect dysregulated pathways with up- and down-regulated genes (bidirectional dysregulation) that are ubiquitous in biological systems. We developed N-of-1-pathways MixEnrich, a mixture model followed by a gene set enrichment test, to uncover bidirectional and concordantly dysregulated pathways one patient at a time. We assess its accuracy in a comprehensive simulation study and in a RNA-Seq data analysis of head and neck squamous cell carcinomas (HNSCCs). In presence of bidirectionally dysregulated genes in the pathway or in presence of high background noise, MixEnrich substantially outperforms previous single-subject transcriptome analysis methods, both in the simulation study and the HNSCCs data analysis (ROC Curves; higher true positive rates; lower false positive rates). Bidirectional and concordant dysregulated pathways uncovered by MixEnrich in each patient largely overlapped with the quasi-gold standard compared to other single-subject and cohort-based transcriptome analyses. The greater performance of MixEnrich presents an advantage over previous methods to meet the promise of providing accurate personal transcriptome analysis to support precision medicine at point of care.

MICROBIAL LABORATORY GUIDANCE MANUAL FOR THE ...

EPA Pesticide Factsheets

The Long-Term 2 Enhanced Surface Water Treatment Rule Laboratory Instruction Manual will be a compilation of all information needed by laboratories and field personnel to collect, analyze, and report the microbiological data required under the rule. The manual will provide laboratories with a single source of information that currently is available from various sources including the latest versions of Methods 1622 and 1623, including all approved, equivalent modifications; the procedures for E.coli methods approved for use under the LT2ESWTR; lists of vendor sources; data recording forms; data reporting requirements; information on the Laboratory Quality Assurance Evaluation Program for the Analysis of Cryptosporidium in Water; and sample collection procedures. Although most of this information is available elsewhere, a single, comprehensive compendium containing this information is needed to aid utilities and laboratories performing the sampling and analysis activities required under the LT2 rule. This manual will serve as an instruction manual for laboratories to use when collecting data for Crypto, E. coli and turbidity.

Analysis of four toxic metals in a single rice seed by matrix solid phase dispersion -inductively coupled plasma mass spectrometry

NASA Astrophysics Data System (ADS)

He, Xiufen; Chen, Lixia; Chen, Xin; Yu, Huamei; Peng, Lixu; Han, Bingjun

2016-12-01

Toxic metals in rice pose great risks to human health. Metal bioaccumulation in rice grains is a criterion of breeding. Rice breeding requires a sensitive method to determine metal content in single rice grains to assist the variety selection. In the present study, four toxic metals of arsenic (As), cadmium (Cd), chromium (Cr) and lead (Pb) in a single rice grain were determined by a simple and rapid method. The developed method is based on matrix solid phase dispersion using multi-wall carbon nanotubes (MWCNTs) as dispersing agent and analyzed by inductively coupled plasma mass spectrometry. The experimental parameters were systematically investigated. The limits of detection (LOD) were 5.0, 0.6, 10 and 2.1 ng g-1 for As, Cd, Cr, and Pb, respectively, with relative standard deviations (n = 6) of <7.7%, demonstrating the good sensitivity and precision of the method. The results of 30 real world rice samples analyzed by this method agreed well with those obtained by the standard microwave digestion. The amount of sample required was reduced approximately 100 fold in comparison with the microwave digestion. The method has a high application potential for other sample matrices and elements with high sensitivity and sample throughput.

Analysis of four toxic metals in a single rice seed by matrix solid phase dispersion -inductively coupled plasma mass spectrometry.

PubMed

He, Xiufen; Chen, Lixia; Chen, Xin; Yu, Huamei; Peng, Lixu; Han, Bingjun

2016-12-06

Toxic metals in rice pose great risks to human health. Metal bioaccumulation in rice grains is a criterion of breeding. Rice breeding requires a sensitive method to determine metal content in single rice grains to assist the variety selection. In the present study, four toxic metals of arsenic (As), cadmium (Cd), chromium (Cr) and lead (Pb) in a single rice grain were determined by a simple and rapid method. The developed method is based on matrix solid phase dispersion using multi-wall carbon nanotubes (MWCNTs) as dispersing agent and analyzed by inductively coupled plasma mass spectrometry. The experimental parameters were systematically investigated. The limits of detection (LOD) were 5.0, 0.6, 10 and 2.1 ng g -1 for As, Cd, Cr, and Pb, respectively, with relative standard deviations (n = 6) of <7.7%, demonstrating the good sensitivity and precision of the method. The results of 30 real world rice samples analyzed by this method agreed well with those obtained by the standard microwave digestion. The amount of sample required was reduced approximately 100 fold in comparison with the microwave digestion. The method has a high application potential for other sample matrices and elements with high sensitivity and sample throughput.

RNA-Seq analysis to capture the transcriptome landscape of a single cell

PubMed Central

Tang, Fuchou; Barbacioru, Catalin; Nordman, Ellen; Xu, Nanlan; Bashkirov, Vladimir I; Lao, Kaiqin; Surani, M. Azim

2013-01-01

We describe here a protocol for digital transcriptome analysis in a single mouse blastomere using a deep sequencing approach. An individual blastomere was first isolated and put into lysate buffer by mouth pipette. Reverse transcription was then performed directly on the whole cell lysate. After this, the free primers were removed by Exonuclease I and a poly(A) tail was added to the 3′ end of the first-strand cDNA by Terminal Deoxynucleotidyl Transferase. Then the single cell cDNAs were amplified by 20 plus 9 cycles of PCR. Then 100-200 ng of these amplified cDNAs were used to construct a sequencing library. The sequencing library can be used for deep sequencing using the SOLiD system. Compared with the cDNA microarray technique, our assay can capture up to 75% more genes expressed in early embryos. The protocol can generate deep sequencing libraries within 6 days for 16 single cell samples. PMID:20203668

Meta-analysis of gene-level tests for rare variant association.

PubMed

Liu, Dajiang J; Peloso, Gina M; Zhan, Xiaowei; Holmen, Oddgeir L; Zawistowski, Matthew; Feng, Shuang; Nikpay, Majid; Auer, Paul L; Goel, Anuj; Zhang, He; Peters, Ulrike; Farrall, Martin; Orho-Melander, Marju; Kooperberg, Charles; McPherson, Ruth; Watkins, Hugh; Willer, Cristen J; Hveem, Kristian; Melander, Olle; Kathiresan, Sekar; Abecasis, Gonçalo R

2014-02-01

The majority of reported complex disease associations for common genetic variants have been identified through meta-analysis, a powerful approach that enables the use of large sample sizes while protecting against common artifacts due to population structure and repeated small-sample analyses sharing individual-level data. As the focus of genetic association studies shifts to rare variants, genes and other functional units are becoming the focus of analysis. Here we propose and evaluate new approaches for performing meta-analysis of rare variant association tests, including burden tests, weighted burden tests, variable-threshold tests and tests that allow variants with opposite effects to be grouped together. We show that our approach retains useful features from single-variant meta-analysis approaches and demonstrate its use in a study of blood lipid levels in ∼18,500 individuals genotyped with exome arrays.

Human leukocyte antigen class I region single-nucleotide polymorphisms are associated with leprosy susceptibility in Vietnam and India.

PubMed

Alter, Andrea; Huong, Nguyen Thu; Singh, Meenakshi; Orlova, Marianna; Van Thuc, Nguyen; Katoch, Kiran; Gao, Xiaojiang; Thai, Vu Hong; Ba, Nguyen Ngoc; Carrington, Mary; Abel, Laurent; Mehra, Narinder; Alcaïs, Alexandre; Schurr, Erwin

2011-05-01

Experimental evidence suggested the existence of unidentified leprosy susceptibility loci in the human leukocyte antigen (HLA) complex. To identify such genetic risk factors, a high-density association scan of a 1.9-mega-base (Mb) region in the HLA complex was performed. Among 682 single-nucleotide polymorphisms (SNPs), 59 were associated with leprosy (P <.01) in 198 Vietnamese single-case leprosy families. Genotyping of these SNPs in an independent sample of 292 Vietnamese single-case leprosy families replicated the association of 12 SNPs (P <.01). Multivariate analysis of these 12 SNPs showed that the association information could be captured by 2 intergenic HLA class I region SNPs (P = 9.4 × 10⁻⁹)-rs2394885 and rs2922997 (marginal multivariate P = 2.1 × 10⁻⁷ and P = .0016, respectively). SNP rs2394885 tagged the HLA-C*15:05 allele in the Vietnamese population. The identical associations were validated in a third sample of 364 patients with leprosy and 371 control subjects from North India. These results implicated class I alleles in leprosy pathogenesis.

Human Leukocyte Antigen Class I Region Single-Nucleotide Polymorphisms are Associated with Leprosy Susceptibility in Vietnam and India

PubMed Central

Alter, Andrea; Huong, Nguyen Thu; Singh, Meenakshi; Orlova, Marianna; Van Thuc, Nguyen; Katoch, Kiran; Gao, Xiaojiang; Thai, Vu Hong; Ba, Nguyen Ngoc; Carrington, Mary; Abel, Laurent; Mehra, Narinder; Alcaïs, Alexandre

2011-01-01

Experimental evidence suggested the existence of unidentified leprosy susceptibility loci in the human leukocyte antigen (HLA) complex. To identify such genetic risk factors, a high-density association scan of a 1.9-mega-base (Mb) region in the HLA complex was performed. Among 682 single-nucleotide polymorphisms (SNPs), 59 were associated with leprosy (P <.01) in 198 Vietnamese single-case leprosy families. Genotyping of these SNPs in an independent sample of 292 Vietnamese single-case leprosy families replicated the association of 12 SNPs (P <.01). Multivariate analysis of these 12 SNPs showed that the association information could be captured by 2 intergenic HLA class I region SNPs (P = 9.4 × 10−9)—rs2394885 and rs2922997 (marginal multivariate P = 2.1 × 10−7 and P = .0016, respectively). SNP rs2394885 tagged the HLA-C*15:05 allele in the Vietnamese population. The identical associations were validated in a third sample of 364 patients with leprosy and 371 control subjects from North India. These results implicated class I alleles in leprosy pathogenesis. PMID:21459816

Categorizing Cells on the Basis of their Chemical Profiles: Progress in Single-Cell Mass Spectrometry.

PubMed

Comi, Troy J; Do, Thanh D; Rubakhin, Stanislav S; Sweedler, Jonathan V

2017-03-22

The chemical differences between individual cells within large cellular populations provide unique information on organisms' homeostasis and the development of diseased states. Even genetically identical cell lineages diverge due to local microenvironments and stochastic processes. The minute sample volumes and low abundance of some constituents in cells hinder our understanding of cellular heterogeneity. Although amplification methods facilitate single-cell genomics and transcriptomics, the characterization of metabolites and proteins remains challenging both because of the lack of effective amplification approaches and the wide diversity in cellular constituents. Mass spectrometry has become an enabling technology for the investigation of individual cellular metabolite profiles with its exquisite sensitivity, large dynamic range, and ability to characterize hundreds to thousands of compounds. While advances in instrumentation have improved figures of merit, acquiring measurements at high throughput and sampling from large populations of cells are still not routine. In this Perspective, we highlight the current trends and progress in mass-spectrometry-based analysis of single cells, with a focus on the technologies that will enable the next generation of single-cell measurements.

Bacterial microbiota of Kazakhstan cheese revealed by single molecule real time (SMRT) sequencing and its comparison with Belgian, Kalmykian and Italian artisanal cheeses.

PubMed

Li, Jing; Zheng, Yi; Xu, Haiyan; Xi, Xiaoxia; Hou, Qiangchuan; Feng, Shuzhen; Wuri, Laga; Bian, Yanfei; Yu, Zhongjie; Kwok, Lai-Yu; Sun, Zhihong; Sun, Tiansong

2017-01-09

In Kazakhstan, traditional artisanal cheeses have a long history and are widely consumed. The unique characteristics of local artisanal cheeses are almost completely preserved. However, their microbial communities have rarely been reported. The current study firstly generated the Single Molecule, Real-Time (SMRT) sequencing bacterial diversity profiles of 6 traditional artisanal cheese samples of Kazakhstan origin, followed by comparatively analyzed the microbiota composition between the current dataset and those from cheeses originated from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy. Across the Kazakhstan cheese samples, a total of 238 bacterial species belonging to 14 phyla and 140 genera were identified. Lactococcus lactis (28.93%), Lactobacillus helveticus (26.43%), Streptococcus thermophilus (12.18%) and Lactobacillus delbrueckii (12.15%) were the dominant bacterial species for these samples. To further evaluate the cheese bacterial diversity of Kazakhstan cheeses in comparison with those from other geographic origins, 16S rRNA datasets of 36 artisanal cheeses from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy were retrieved from public databases. The cheese bacterial microbiota communities were largely different across sample origins. By principal coordinate analysis (PCoA) and multivariate analysis of variance (MANOVA), the structure of the Kazakhstan artisanal cheese samples was found to be different from those of the other geographic origins. Furthermore, the redundancy analysis (RDA) identified 16 bacterial OTUs as the key variables responsible for such microbiota structural difference. Our results together suggest that the diversity of bacterial communities in different groups is stratified by geographic region. This study does not only provide novel information on the bacterial microbiota of traditional artisanal cheese of Kazakhstan at species level, but also interesting insights into the bacterial diversity of artisanal cheeses of various geographical origins.

Single-particle investigation of summertime and wintertime Antarctic sea spray aerosols using low-Z particle EPMA, Raman microspectrometry, and ATR-FTIR imaging techniques

NASA Astrophysics Data System (ADS)

Eom, Hyo-Jin; Gupta, Dhrubajyoti; Cho, Hye-Rin; Hwang, Hee Jin; Do Hur, Soon; Gim, Yeontae; Ro, Chul-Un

2016-11-01

Two aerosol samples collected at King Sejong Korean scientific research station, Antarctica, on 9 December 2011 in the austral summer (sample S1) and 23 July 2012 in the austral winter (sample S2), when the oceanic chlorophyll a levels on the collection days of the samples were quite different, by ˜ 19 times (2.46 vs. 0.13 µg L-1, respectively), were investigated on a single-particle basis using quantitative energy-dispersive electron probe X-ray microanalysis (ED-EPMA), called low-Z particle EPMA, Raman microspectrometry (RMS), and attenuated total reflection Fourier transform infrared (ATR-FTIR) imaging techniques to obtain their characteristics based on the elemental chemical compositions, molecular species, and mixing state. X-ray analysis showed that the supermicron summertime and wintertime Antarctic aerosol samples have different elemental chemical compositions, even though all the individual particles analyzed were sea spray aerosols (SSAs); i.e., the contents of C, O, Ca, S, and Si were more elevated, whereas Cl was more depleted, for sample S1 than for sample S2. Based on qualitative analysis of the chemical species present in individual SSAs by the combined application of RMS and ATR-FTIR imaging, different organic species were observed in samples S1 and S2; i.e., Mg hydrate salts of alanine were predominant in samples S1 and S2, whereas Mg salts of fatty acids internally mixed with Mg hydrate salts of alanine were significant in sample S2. Although CaSO4 was observed significantly in both samples S1 and S2, other inorganic species, such as Na2SO4, NaNO3, Mg(NO3)2, SiO2, and CH3SO3Mg, were observed more significantly in sample S1, suggesting that those compounds may be related to the higher phytoplankton activity in summer.

Determination of the botanical origin of honey by front-face synchronous fluorescence spectroscopy.

PubMed

Lenhardt, Lea; Zeković, Ivana; Dramićanin, Tatjana; Dramićanin, Miroslav D; Bro, Rasmus

2014-01-01

Front-face synchronous fluorescence spectroscopy combined with chemometrics is used to classify honey samples according to their botanical origin. Synchronous fluorescence spectra of three monofloral (linden, sunflower, and acacia), polyfloral (meadow mix), and fake (fake acacia and linden) honey types (109 samples) were collected in an excitation range of 240-500 nm for synchronous wavelength intervals of 30-300 nm. Chemometric analysis of the gathered data included principal component analysis and partial least squares discriminant analysis. Mean cross-validated classification errors of 0.2 and 4.8% were found for a model that accounts only for monofloral samples and for a model that includes both the monofloral and polyfloral groups, respectively. The results demonstrate that single synchronous fluorescence spectra of different honeys differ significantly because of their distinct physical and chemical characteristics and provide sufficient data for the clear differentiation among honey groups. The spectra of fake honey samples showed pronounced differences from those of genuine honey, and these samples are easily recognized on the basis of their synchronous fluorescence spectra. The study demonstrated that this method is a valuable and promising technique for honey authentication.

Classical test theory and Rasch analysis validation of the Upper Limb Functional Index in subjects with upper limb musculoskeletal disorders.

PubMed

Bravini, Elisabetta; Franchignoni, Franco; Giordano, Andrea; Sartorio, Francesco; Ferriero, Giorgio; Vercelli, Stefano; Foti, Calogero

2015-01-01

To perform a comprehensive analysis of the psychometric properties and dimensionality of the Upper Limb Functional Index (ULFI) using both classical test theory and Rasch analysis (RA). Prospective, single-group observational design. Freestanding rehabilitation center. Convenience sample of Italian-speaking subjects with upper limb musculoskeletal disorders (N=174). Not applicable. The Italian version of the ULFI. Data were analyzed using parallel analysis, exploratory factor analysis, and RA for evaluating dimensionality, functioning of rating scale categories, item fit, hierarchy of item difficulties, and reliability indices. Parallel analysis revealed 2 factors explaining 32.5% and 10.7% of the response variance. RA confirmed the failure of the unidimensionality assumption, and 6 items out of the 25 misfitted the Rasch model. When the analysis was rerun excluding the misfitting items, the scale showed acceptable fit values, loading meaningfully to a single factor. Item separation reliability and person separation reliability were .98 and .89, respectively. Cronbach alpha was .92. RA revealed weakness of the scale concerning dimensionality and internal construct validity. However, a set of 19 ULFI items defined through the statistical process demonstrated a unidimensional structure, good psychometric properties, and clinical meaningfulness. These findings represent a useful starting point for further analyses of the tool (based on modern psychometric approaches and confirmatory factor analysis) in larger samples, including different patient populations and nationalities. Copyright © 2015 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.

Noninvasive prenatal diagnosis for single gene disorders.

PubMed

Allen, Stephanie; Young, Elizabeth; Bowns, Benjamin

2017-04-01

Noninvasive prenatal diagnosis for single gene disorders is coming to fruition in its clinical utility. The presence of cell-free DNA in maternal plasma has been recognized for many years, and a number of applications have developed from this. Noninvasive prenatal diagnosis for single gene disorders has lagged behind due to complexities of technology development, lack of investment and the need for validation samples for rare disorders. Publications are emerging demonstrating a variety of technical approaches and feasibility of clinical application. Techniques for analysis of cell-free DNA including digital PCR, next-generation sequencing and relative haplotype dosage have been used most often for assay development. Analysis of circulating fetal cells in the maternal blood is still being investigated as a viable alternative and more recently transcervical trophoblast cells. Studies exploring ethical and social issues are generally positive but raise concerns around the routinization of prenatal testing. Further work is necessary to make testing available to all patients with a pregnancy at risk of a single gene disorder, and it remains to be seen if the development of more powerful technologies such as isolation and analysis of single cells will shift the emphasis of noninvasive prenatal diagnosis. As testing becomes possible for a wider range of conditions, more ethical questions will become relevant.

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

PubMed

Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund

2017-05-31

Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

Direct analysis of hCGβcf glycosylation in normal and aberrant pregnancy by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Iles, Ray K; Cole, Laurence A; Butler, Stephen A

2014-06-05

The analysis of human chorionic gonadotropin (hCG) in clinical chemistry laboratories by specific immunoassay is well established. However, changes in glycosylation are not as easily assayed and yet alterations in hCG glycosylation is associated with abnormal pregnancy. hCGβ-core fragment (hCGβcf) was isolated from the urine of women, pregnant with normal, molar and hyperemesis gravidarum pregnancies. Each sample was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis following dithiothreitol (DTT) reduction and fingerprint spectra of peptide hCGβ 6-40 were analyzed. Samples were variably glycosylated, where most structures were small, core and largely mono-antennary. Larger single bi-antennary and mixtures of larger mono-antennary and bi-antennary moieties were also observed in some samples. Larger glycoforms were more abundant in the abnormal pregnancies and tri-antennary carbohydrate moieties were only observed in the samples from molar and hyperemesis gravidarum pregnancies. Given that such spectral profiling differences may be characteristic, development of small sample preparation for mass spectral analysis of hCG may lead to a simpler and faster approach to glycostructural analysis and potentially a novel clinical diagnostic test.

Whole-genome analysis of mycobacteria from birds at the San Diego Zoo.

PubMed

Pfeiffer, Wayne; Braun, Josephine; Burchell, Jennifer; Witte, Carmel L; Rideout, Bruce A

2017-01-01

Mycobacteria isolated from more than 100 birds diagnosed with avian mycobacteriosis at the San Diego Zoo and its Safari Park were cultured postmortem and had their whole genomes sequenced. Computational workflows were developed and applied to identify the mycobacterial species in each DNA sample, to find single-nucleotide polymorphisms (SNPs) between samples of the same species, to further differentiate SNPs between as many as three different genotypes within a single sample, and to identify which samples are closely clustered genomically. Nine species of mycobacteria were found in 123 samples from 105 birds. The most common species were Mycobacterium avium and Mycobacterium genavense, which were in 49 and 48 birds, respectively. Most birds contained only a single mycobacterial species, but two birds contained a mixture of two species. The M. avium samples represent diverse strains of M. avium avium and M. avium hominissuis, with many pairs of samples differing by hundreds or thousands of SNPs across their common genome. By contrast, the M. genavense samples are much closer genomically; samples from 46 of 48 birds differ from each other by less than 110 SNPs. Some birds contained two, three, or even four genotypes of the same bacterial species. Such infections were found in 4 of 49 birds (8%) with M. avium and in 11 of 48 birds (23%) with M. genavense. Most were mixed infections, in which the bird was infected by multiple mycobacterial strains, but three infections with two genotypes differing by ≤ 10 SNPs were likely the result of within-host evolution. The samples from 31 birds with M. avium can be grouped into nine clusters within which any sample is ≤ 12 SNPs from at least one other sample in the cluster. Similarly, the samples from 40 birds with M. genavense can be grouped into ten such clusters. Information about these genomic clusters is being used in an ongoing, companion study of mycobacterial transmission to help inform management of bird collections.

Whole-genome analysis of mycobacteria from birds at the San Diego Zoo

PubMed Central

Pfeiffer, Wayne; Braun, Josephine; Burchell, Jennifer; Witte, Carmel L.; Rideout, Bruce A.

2017-01-01

Methods Mycobacteria isolated from more than 100 birds diagnosed with avian mycobacteriosis at the San Diego Zoo and its Safari Park were cultured postmortem and had their whole genomes sequenced. Computational workflows were developed and applied to identify the mycobacterial species in each DNA sample, to find single-nucleotide polymorphisms (SNPs) between samples of the same species, to further differentiate SNPs between as many as three different genotypes within a single sample, and to identify which samples are closely clustered genomically. Results Nine species of mycobacteria were found in 123 samples from 105 birds. The most common species were Mycobacterium avium and Mycobacterium genavense, which were in 49 and 48 birds, respectively. Most birds contained only a single mycobacterial species, but two birds contained a mixture of two species. The M. avium samples represent diverse strains of M. avium avium and M. avium hominissuis, with many pairs of samples differing by hundreds or thousands of SNPs across their common genome. By contrast, the M. genavense samples are much closer genomically; samples from 46 of 48 birds differ from each other by less than 110 SNPs. Some birds contained two, three, or even four genotypes of the same bacterial species. Such infections were found in 4 of 49 birds (8%) with M. avium and in 11 of 48 birds (23%) with M. genavense. Most were mixed infections, in which the bird was infected by multiple mycobacterial strains, but three infections with two genotypes differing by ≤ 10 SNPs were likely the result of within-host evolution. The samples from 31 birds with M. avium can be grouped into nine clusters within which any sample is ≤ 12 SNPs from at least one other sample in the cluster. Similarly, the samples from 40 birds with M. genavense can be grouped into ten such clusters. Information about these genomic clusters is being used in an ongoing, companion study of mycobacterial transmission to help inform management of bird collections. PMID:28267758

Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq

PubMed Central

Loeffler-Wirth, Henry; Hopp, Lydia; Schadendorf, Dirk; Schartl, Manfred; Anderegg, Ulf; Camp, Gray; Treutlein, Barbara; Binder, Hans; Kunz, Manfred

2017-01-01

Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy. PMID:27903987

Thermal analysis of Bridgman-Stockbarger growth. [mercury cadmium telluride single crystals

NASA Technical Reports Server (NTRS)

Knopf, F. W.

1979-01-01

A thermal analysis of a cylindrical HgCdTe sample in a Bridgman-Stockbarger crystal growth configuration was conducted with emphasis on the thermal profile, interface shape and position, and the thermal gradients at the liquid-solid interface. Alloys of HgTe and CdTe with compositions approximating 20 percent CdTe, 80 percent HgTe were used. This composition results in a bandgap suited for the detection of 10.6 micron CO2 radiation. The sensitivity of the sample thermal characteristics to important growth parameters, such as thermal diffusivities, thermal conductivities, furnace temperature profile, ampoule dimensions, and growth velocity was assessed. Numerical techniques and associated computational models necessary to analyze the heat transfer process within the sample and the Bridgman-Stockbarger boundary conditions were developed. This thermal analysis mode was programmed in FORTRAN V, and is currently operational on the MSFC Univac 1100 system.

Comparison of passive diffusion bag samplers and submersible pump sampling methods for monitoring volatile organic compounds in ground water at Area 6, Naval Air Station, Whidbey Island, Washington

USGS Publications Warehouse

Huffman, Raegan L.

2002-01-01

Ground-water samples were collected in April 1999 at Naval Air Station Whidbey Island, Washington, with passive diffusion samplers and a submersible pump to compare concentrations of volatile organic compounds (VOCs) in water samples collected using the two sampling methods. Single diffusion samplers were installed in wells with 10-foot screened intervals, and multiple diffusion samplers were installed in wells with 20- to 40-foot screened intervals. The diffusion samplers were recovered after 20 days and the wells were then sampled using a submersible pump. VOC concentrations in the 10-foot screened wells in water samples collected with diffusion samplers closely matched concentrations in samples collected with the submersible pump. Analysis of VOC concentrations in samples collected from the 20- to 40-foot screened wells with multiple diffusion samplers indicated vertical concentration variation within the screened interval, whereas the analysis of VOC concentrations in samples collected with the submersible pump indicated mixing during pumping. The results obtained using the two sampling methods indicate that the samples collected with the diffusion samplers were comparable with and can be considerably less expensive than samples collected using a submersible pump.

Rapid quantification and sex determination of forensic evidence materials.

PubMed

Andréasson, Hanna; Allen, Marie

2003-11-01

DNA quantification of forensic evidence is very valuable for an optimal use of the available biological material. Moreover, sex determination is of great importance as additional information in criminal investigations as well as in identification of missing persons, no suspect cases, and ancient DNA studies. While routine forensic DNA analysis based on short tandem repeat markers includes a marker for sex determination, analysis of samples containing scarce amounts of DNA is often based on mitochondrial DNA, and sex determination is not performed. In order to allow quantification and simultaneous sex determination on minute amounts of DNA, an assay based on real-time PCR analysis of a marker within the human amelogenin gene has been developed. The sex determination is based on melting curve analysis, while an externally standardized kinetic analysis allows quantification of the nuclear DNA copy number in the sample. This real-time DNA quantification assay has proven to be highly sensitive, enabling quantification of single DNA copies. Although certain limitations were apparent, the system is a rapid, cost-effective, and flexible assay for analysis of forensic casework samples.

7 CFR 43.104 - Master table of single and double sampling plans.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Master table of single and double sampling plans. 43... STANDARD CONTAINER REGULATIONS STANDARDS FOR SAMPLING PLANS Sampling Plans § 43.104 Master table of single and double sampling plans. (a) In the master table, a sampling plan is selected by first determining...

7 CFR 43.104 - Master table of single and double sampling plans.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Master table of single and double sampling plans. 43... STANDARD CONTAINER REGULATIONS STANDARDS FOR SAMPLING PLANS Sampling Plans § 43.104 Master table of single and double sampling plans. (a) In the master table, a sampling plan is selected by first determining...

7 CFR 43.104 - Master table of single and double sampling plans.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Master table of single and double sampling plans. 43... STANDARD CONTAINER REGULATIONS STANDARDS FOR SAMPLING PLANS Sampling Plans § 43.104 Master table of single and double sampling plans. (a) In the master table, a sampling plan is selected by first determining...

7 CFR 43.104 - Master table of single and double sampling plans.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Master table of single and double sampling plans. 43... STANDARD CONTAINER REGULATIONS STANDARDS FOR SAMPLING PLANS Sampling Plans § 43.104 Master table of single and double sampling plans. (a) In the master table, a sampling plan is selected by first determining...

Quantitative DNA Methylation Analysis Identifies a Single CpG Dinucleotide Important for ZAP-70 Expression and Predictive of Prognosis in Chronic Lymphocytic Leukemia

PubMed Central

Claus, Rainer; Lucas, David M.; Stilgenbauer, Stephan; Ruppert, Amy S.; Yu, Lianbo; Zucknick, Manuela; Mertens, Daniel; Bühler, Andreas; Oakes, Christopher C.; Larson, Richard A.; Kay, Neil E.; Jelinek, Diane F.; Kipps, Thomas J.; Rassenti, Laura Z.; Gribben, John G.; Döhner, Hartmut; Heerema, Nyla A.; Marcucci, Guido; Plass, Christoph; Byrd, John C.

2012-01-01

Purpose Increased ZAP-70 expression predicts poor prognosis in chronic lymphocytic leukemia (CLL). Current methods for accurately measuring ZAP-70 expression are problematic, preventing widespread application of these tests in clinical decision making. We therefore used comprehensive DNA methylation profiling of the ZAP-70 regulatory region to identify sites important for transcriptional control. Patients and Methods High-resolution quantitative DNA methylation analysis of the entire ZAP-70 gene regulatory regions was conducted on 247 samples from patients with CLL from four independent clinical studies. Results Through this comprehensive analysis, we identified a small area in the 5′ regulatory region of ZAP-70 that showed large variability in methylation in CLL samples but was universally methylated in normal B cells. High correlation with mRNA and protein expression, as well as activity in promoter reporter assays, revealed that within this differentially methylated region, a single CpG dinucleotide and neighboring nucleotides are particularly important in ZAP-70 transcriptional regulation. Furthermore, by using clustering approaches, we identified a prognostic role for this site in four independent data sets of patients with CLL using time to treatment, progression-free survival, and overall survival as clinical end points. Conclusion Comprehensive quantitative DNA methylation analysis of the ZAP-70 gene in CLL identified important regions responsible for transcriptional regulation. In addition, loss of methylation at a specific single CpG dinucleotide in the ZAP-70 5′ regulatory sequence is a highly predictive and reproducible biomarker of poor prognosis in this disease. This work demonstrates the feasibility of using quantitative specific ZAP-70 methylation analysis as a relevant clinically applicable prognostic test in CLL. PMID:22564988

A proposed method to minimize waste from institutional radiation safety surveillance programs through the application of expected value statistics.

PubMed

Emery, R J

1997-03-01

Institutional radiation safety programs routinely use wipe test sampling and liquid scintillation counting analysis to indicate the presence of removable radioactive contamination. Significant volumes of liquid waste can be generated by such surveillance activities, and the subsequent disposal of these materials can sometimes be difficult and costly. In settings where large numbers of negative results are regularly obtained, the limited grouping of samples for analysis based on expected value statistical techniques is possible. To demonstrate the plausibility of the approach, single wipe samples exposed to varying amounts of contamination were analyzed concurrently with nine non-contaminated samples. Although the sample grouping inevitably leads to increased quenching with liquid scintillation counting systems, the effect did not impact the ability to detect removable contamination in amounts well below recommended action levels. Opportunities to further improve this cost effective semi-quantitative screening procedure are described, including improvements in sample collection procedures, enhancing sample-counting media contact through mixing and extending elution periods, increasing sample counting times, and adjusting institutional action levels.

Plural output optimetric sample cell and analysis system

NASA Technical Reports Server (NTRS)

Haley, F. C. (Inventor)

1971-01-01

An apparatus suitable for receiving a sample for optimetric analysis includes a sample cell comprising an opaque hollow tube. Several apertures are defined in the wall of the tubing and a lens barrel which extends beyond to opposite surfaces of the wall is supported within at least one of the apertures. A housing is provided with one channel for receiving the sample cell and a series of channels extending from the exterior housing to the sample cell apertures. A filter element is housed in each of these latter channels. These channels slidingly receive an excitation light source for a photodetector cell to permit selective focusing. A sample cell containing at least three apertures in the walls can be mounted for rotation relative to a light source or photoconduction means for simultaneous or alternative optimetric determination of the components of a single sample. The sample cell is fabricated by supporting a lens barrel within the aperture. A molten portion of glass is deposited in the lens barrel and cooled while in a horizontal position to form a lens having an acceptable angle.

Uncertainty in monitoring E. coli concentrations in streams and stormwater runoff

NASA Astrophysics Data System (ADS)

Harmel, R. D.; Hathaway, J. M.; Wagner, K. L.; Wolfe, J. E.; Karthikeyan, R.; Francesconi, W.; McCarthy, D. T.

2016-03-01

Microbial contamination of surface waters, a substantial public health concern throughout the world, is typically identified by fecal indicator bacteria such as Escherichia coli. Thus, monitoring E. coli concentrations is critical to evaluate current conditions, determine restoration effectiveness, and inform model development and calibration. An often overlooked component of these monitoring and modeling activities is understanding the inherent random and systematic uncertainty present in measured data. In this research, a review and subsequent analysis was performed to identify, document, and analyze measurement uncertainty of E. coli data collected in stream flow and stormwater runoff as individual discrete samples or throughout a single runoff event. Data on the uncertainty contributed by sample collection, sample preservation/storage, and laboratory analysis in measured E. coli concentrations were compiled and analyzed, and differences in sampling method and data quality scenarios were compared. The analysis showed that: (1) manual integrated sampling produced the lowest random and systematic uncertainty in individual samples, but automated sampling typically produced the lowest uncertainty when sampling throughout runoff events; (2) sample collection procedures often contributed the highest amount of uncertainty, although laboratory analysis introduced substantial random uncertainty and preservation/storage introduced substantial systematic uncertainty under some scenarios; and (3) the uncertainty in measured E. coli concentrations was greater than that of sediment and nutrients, but the difference was not as great as may be assumed. This comprehensive analysis of uncertainty in E. coli concentrations measured in streamflow and runoff should provide valuable insight for designing E. coli monitoring projects, reducing uncertainty in quality assurance efforts, regulatory and policy decision making, and fate and transport modeling.

High pressure single-crystal micro X-ray diffraction analysis with GSE_ADA/RSV software

NASA Astrophysics Data System (ADS)

Dera, Przemyslaw; Zhuravlev, Kirill; Prakapenka, Vitali; Rivers, Mark L.; Finkelstein, Gregory J.; Grubor-Urosevic, Ognjen; Tschauner, Oliver; Clark, Simon M.; Downs, Robert T.

2013-08-01

GSE_ADA/RSV is a free software package for custom analysis of single-crystal micro X-ray diffraction (SCμXRD) data, developed with particular emphasis on data from samples enclosed in diamond anvil cells and subject to high pressure conditions. The package has been in extensive use at the high pressure beamlines of Advanced Photon Source (APS), Argonne National Laboratory and Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. The software is optimized for processing of wide-rotation images and includes a variety of peak intensity corrections and peak filtering features, which are custom-designed to make processing of high pressure SCμXRD easier and more reliable.

Annotate-it: a Swiss-knife approach to annotation, analysis and interpretation of single nucleotide variation in human disease

PubMed Central

2012-01-01

The increasing size and complexity of exome/genome sequencing data requires new tools for clinical geneticists to discover disease-causing variants. Bottlenecks in identifying the causative variation include poor cross-sample querying, constantly changing functional annotation and not considering existing knowledge concerning the phenotype. We describe a methodology that facilitates exploration of patient sequencing data towards identification of causal variants under different genetic hypotheses. Annotate-it facilitates handling, analysis and interpretation of high-throughput single nucleotide variant data. We demonstrate our strategy using three case studies. Annotate-it is freely available and test data are accessible to all users at http://www.annotate-it.org. PMID:23013645

Iron single crystal growth from a lithium-rich melt

NASA Astrophysics Data System (ADS)

Fix, M.; Schumann, H.; Jantz, S. G.; Breitner, F. A.; Leineweber, A.; Jesche, A.

2018-03-01

α -Fe single crystals of rhombic dodecahedral habit were grown from a Li84N12Fe∼3 melt. Crystals of several millimeter along a side form at temperatures around T ≈ 800 ° C. Upon further cooling the growth competes with the formation of Fe-doped Li3N. The b.c.c. structure and good sample quality of α -Fe single crystals were confirmed by X-ray and electron diffraction as well as magnetization measurements and chemical analysis. A nitrogen concentration of 90 ppm was detected by means of carrier gas hot extraction. Scanning electron microscopy did not reveal any sign of iron nitride precipitates.

Development of a single-axis ultrasonic levitator and the study of the radial particle oscillations

NASA Astrophysics Data System (ADS)

Baer, Sebastian; Andrade, Marco A. B.; Esen, Cemal; Adamowski, Julio Cezar; Ostendorf, Andreas

2012-05-01

This work describes the development and analysis of a new single-axis acoustic levitator, which consists of a 38 kHz Langevin-type piezoelectric transducer with a concave radiating surface and a concave reflector. The new levitator design allows to significantly reducing the electric power necessary to levitate particles and to stabilize the levitated sample in both radial and axial directions. In this investigation the lateral oscillations of a levitated particle were measured with a single point Laser Doppler Vibrometer (LDV) and an image evaluation technique. The lateral oscillations were measured for different values of particle diameter, particle density and applied electrical power.

1000540

NASA Image and Video Library

2010-04-13

ERIC EARHART (AEROSPACE ENGINEER, ER41 PROPULSION STRUCTURAL & DYNAMICS ANALYSIS BRANCH) DISCUSSES DATA PRODUCED BY A UNIQUE MECHANICAL TEST SETUP THAT MEASURES STRAIN ON A SINGLE SAMPLE, USING TWO DIFFERENT TECHNIQUES AT THE SAME TIME. THE TEST FIXTURE HOLDS A SPECIMEN THAT REPRESENTS A LIQUID OXYGEN (LOX) BEARING FROM THE J2-X ENGINE

Interleukin 1 beta gene and risk of schizophrenia: detailed case-control and family-based studies and an updated meta-analysis.

PubMed

Shibuya, Masako; Watanabe, Yuichiro; Nunokawa, Ayako; Egawa, Jun; Kaneko, Naoshi; Igeta, Hirofumi; Someya, Toshiyuki

2014-01-01

Interleukin-1 beta (IL-1β) has been implicated in the pathophysiology of schizophrenia. To assess whether the IL1B gene confers increased susceptibility to schizophrenia, we conducted case-control and family-based studies and an updated meta-analysis. We tested the association between IL1B and schizophrenia in 1229 case-control and 112 trio samples using 12 markers, including common tagging single nucleotide variations (SNVs) and a rare non-synonymous variation detected by resequencing the coding regions. We also performed a meta-analysis of rs16944 using a total of 8724 case-control and 201 trio samples from 16 independent populations. We found no significant associations between any of the 12 SNVs examined and schizophrenia in either case-control or trio samples. Moreover, our meta-analysis results showed no significant association between the common SNV, rs16944, and schizophrenia. The present study does not support a role for IL1B in schizophrenia susceptibility.

LC-QTOF-MS Analysis and Activity Profiles of Popular Antioxidant Dietary Supplements in Terms of Quality Control

PubMed Central

Baj, Tomasz; Sawicki, Rafal; Wanat, Aleksandra; Wojtanowski, Krzysztof Kamil; Ginalska, Grazyna; Zgorka, Grazyna; Szymanska, Jolanta

2017-01-01

The dietary supplements with claimed antioxidant activity constitute a substantial part of the dietary supplement market. In this study, we performed the LC-QTOF-MS analysis and investigated the activity profiles of popular antioxidant dietary supplements from different chemical groups in terms of quality control. The commonly used antioxidant tests and statistical analysis revealed that substantial part of the results was comparable if 1 g sample was considered, but while comparing single and daily doses, significant differences in antioxidant values were noticed in all assays. The best antioxidant activity was obtained in ORAC assay (from 142 to 13814 μM of Trolox equivalents per 1 g of sample), and the strongest correlation occurred between TPC and ORAC. The LC-QTOF-MS analysis revealed that catechins were present in samples having the best antioxidant activity and that dietary supplements showing the weakest activity contained very small amount of any chemical constituents. PMID:28642814

Noninvasive analysis of microbiome dynamics in the fruit fly Drosophila melanogaster.

PubMed

Fink, Christine; Staubach, Fabian; Kuenzel, Sven; Baines, John F; Roeder, Thomas

2013-11-01

The diversity and structure of the intestinal microbial community has a strong influence on life history. To understand how hosts and microbes interact, model organisms with comparatively simple microbial communities, such as the fruit fly (Drosophila melanogaster), offer key advantages. However, studies of the Drosophila microbiome are limited to a single point in time, because flies are typically sacrificed for DNA extraction. In order to test whether noninvasive approaches, such as sampling of fly feces, could be a means to assess fly-associated communities over time on the same cohort of flies, we compared the microbial communities of fly feces, dissected fly intestines, and whole flies across three different Drosophila strains. Bacterial species identified in either whole flies or isolated intestines were reproducibly found in feces samples. Although the bacterial communities of feces and intestinal samples were not identical, they shared similarities and obviously the same origin. In contrast to material from whole flies and intestines, feces samples were not compromised by Wolbachia spp. infections, which are widespread in laboratory and wild strains. In a proof-of-principle experiment, we showed that simple nutritional interventions, such as a high-fat diet or short-term starvation, had drastic and long-lasting effects on the micobiome. Thus, the analysis of feces can supplement the toolbox for microbiome studies in Drosophila, unleashing the full potential of such studies in time course experiments where multiple samples from single populations are obtained during aging, development, or experimental manipulations.

Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.

PubMed

Kristensen, Lasse S; Wojdacz, Tomasz K; Thestrup, Britta B; Wiuf, Carsten; Hager, Henrik; Hansen, Lise Lotte

2009-12-21

The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. MS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.

Determination of linuron and related compounds in soil by microwave-assisted solvent extraction and reversed-phase liquid chromatography with UV detection.

PubMed

Molins, C; Hogendoorn, E A; Dijkman, E; Heusinkveld, H A; Baumann, R A

2000-02-11

The combination of microwave-assisted solvent extraction (MASE) and reversed-phase liquid chromatography (RPLC) with UV detection has been investigated for the efficient determination of phenylurea herbicides in soils involving the single-residue method (SRM) approach (linuron) and the multi-residue method (MRM) approach (monuron, monolinuron, isoproturon, metobromuron, diuron and linuron). Critical parameters of MASE, viz, extraction temperature, water content and extraction solvent were varied in order to optimise recoveries of the analytes while simultaneously minimising co-extraction of soil interferences. The optimised extraction procedure was applied to different types of soil with an organic carbon content of 0.4-16.7%. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. A comparative study between the applicability of RPLC-UV without and with the use of column switching for the processing of uncleaned extracts, was carried out. For some of the tested analyte/matrix combinations the one-column approach (LC mode) is feasible. In comparison to LC, coupled-column LC (LC-LC mode) provides high selectivity in single-residue analysis (linuron) and, although less pronounced in multi-residue analysis (all six phenylurea herbicides), the clean-up performance of LC-LC improves both time of analysis and sample throughput. In the MRM approach the developed procedure involving MASE and LC-LC-UV provided acceptable recoveries (range, 80-120%) and RSDs (<12%) at levels of 10 microg/kg (n=9) and 50 microg/kg (n=7), respectively, for most analyte/matrix combinations. Recoveries from aged residue samples spiked at a level of 100 microg/kg (n=7) ranged, depending of the analyte/soil type combination, from 41-113% with RSDs ranging from 1-35%. In the SRM approach the developed LC-LC procedure was applied for the determination of linuron in 28 sandy soil samples collected in a field study. Linuron could be determined in soil with a limit of quantitation of 10 microg/kg.

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi).

PubMed

Gaydos, J K; Miller, W A; Johnson, C; Zornetzer, H; Melli, A; Packham, A; Jeffries, S J; Lance, M M; Conrad, P A

2008-12-01

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.

A comparative study of mid-infrared diffuse reflection (DR) and attenuated total reflection (ATR) spectroscopy for the detection of fungal infection on RWA2-corn.

PubMed

Kos, Gregor; Krska, Rudolf; Lohninger, Hans; Griffiths, Peter R

2004-01-01

An investigation into the rapid detection of mycotoxin-producing fungi on corn by two mid-infrared spectroscopic techniques was undertaken. Corn samples from a single genotype (RWA2, blanks, and contaminated with Fusarium graminearum) were ground, sieved and, after appropriate sample preparation, subjected to mid-infrared spectroscopy using two different accessories (diffuse reflection and attenuated total reflection). The measured spectra were evaluated with principal component analysis (PCA) and the blank and contaminated samples were classified by cluster analysis. Reference data for fungal metabolites were obtained with conventional methods. After extraction and clean-up, each sample was analyzed for the toxin deoxynivalenol (DON) by gas chromatography with electron capture detection (GC-ECD) and ergosterol (a parameter for the total fungal biomass) by high-performance liquid chromatography with diode array detection (HPLC-DAD). The concentration ranges for contaminated samples were 880-3600 microg/kg for ergosterol and 300-2600 microg/kg for DON. Classification efficiency was 100% for ATR spectra. DR spectra did not show as obvious a clustering of contaminated and blank samples. Results and trends were also observed in single spectra plots. Quantification using a PLS1 regression algorithm showed good correlation with DON reference data, but a rather high standard error of prediction (SEP) with 600 microg/kg (DR) and 490 microg/kg (ATR), respectively, for ergosterol. Comparing measurement procedures and results showed advantages for the ATR technique, mainly owing to its ease of use and the easier interpretation of results that were better with respect to classification and quantification.

bigSCale: an analytical framework for big-scale single-cell data.

PubMed

Iacono, Giovanni; Mereu, Elisabetta; Guillaumet-Adkins, Amy; Corominas, Roser; Cuscó, Ivon; Rodríguez-Esteban, Gustavo; Gut, Marta; Pérez-Jurado, Luis Alberto; Gut, Ivo; Heyn, Holger

2018-06-01

Single-cell RNA sequencing (scRNA-seq) has significantly deepened our insights into complex tissues, with the latest techniques capable of processing tens of thousands of cells simultaneously. Analyzing increasing numbers of cells, however, generates extremely large data sets, extending processing time and challenging computing resources. Current scRNA-seq analysis tools are not designed to interrogate large data sets and often lack sensitivity to identify marker genes. With bigSCale, we provide a scalable analytical framework to analyze millions of cells, which addresses the challenges associated with large data sets. To handle the noise and sparsity of scRNA-seq data, bigSCale uses large sample sizes to estimate an accurate numerical model of noise. The framework further includes modules for differential expression analysis, cell clustering, and marker identification. A directed convolution strategy allows processing of extremely large data sets, while preserving transcript information from individual cells. We evaluated the performance of bigSCale using both a biological model of aberrant gene expression in patient-derived neuronal progenitor cells and simulated data sets, which underlines the speed and accuracy in differential expression analysis. To test its applicability for large data sets, we applied bigSCale to assess 1.3 million cells from the mouse developing forebrain. Its directed down-sampling strategy accumulates information from single cells into index cell transcriptomes, thereby defining cellular clusters with improved resolution. Accordingly, index cell clusters identified rare populations, such as reelin ( Reln )-positive Cajal-Retzius neurons, for which we report previously unrecognized heterogeneity associated with distinct differentiation stages, spatial organization, and cellular function. Together, bigSCale presents a solution to address future challenges of large single-cell data sets. © 2018 Iacono et al.; Published by Cold Spring Harbor Laboratory Press.

Determination of the chemical parameters and manufacturer of divins from their broadband transmission spectra

NASA Astrophysics Data System (ADS)

Khodasevich, M. A.; Sinitsyn, G. V.; Skorbanova, E. A.; Rogovaya, M. V.; Kambur, E. I.; Aseev, V. A.

2016-06-01

Analysis of multiparametric data on transmission spectra of 24 divins (Moldovan cognacs) in the 190-2600 nm range allows identification of outliers and their removal from a sample under study in the following consideration. The principal component analysis and classification tree with a single-rank predictor constructed in the 2D space of principal components allow classification of divin manufacturers. It is shown that the accuracy of syringaldehyde, ethyl acetate, vanillin, and gallic acid concentrations in divins calculated with the regression to latent structures depends on the sample volume and is 3, 6, 16, and 20%, respectively, which is acceptable for the application.

Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis.

PubMed

Chang, Chia-Hao; Mau-Hsu, Daxen; Chen, Ke-Cheng; Wei, Cheng-Wey; Chiu, Chiung-Ying; Young, Tai-Horng

2018-02-21

In a single-cell study, isolating and identifying single cells are essential, but these processes often require a large investment of time or money. The aim of this study was to isolate and analyse single cells using a novel platform, the PanelChip™ Analysis System, which includes 2500 microwells chip and a digital real-time polymerase chain reaction (dqPCR) assay, in comparison with a standard PCR (qPCR) assay. Through the serial dilution of a known concentration standard, namely pUC19, the accuracy and sensitivity levels of two methodologies were compared. The two systems were tested on the basis of expression levels of the genetic markers vimentin, E-cadherin, N-cadherin and GAPDH in A549 lung carcinoma cells at two known concentrations. Furthermore, the influence of a known PCR inhibitor commonly found in blood samples, heparin, was evaluated in both methodologies. Finally, mathematical models were proposed and separation method of single cells was verified; moreover, gene expression levels during epithelial-mesenchymal transition in single cells under TGFβ1 treatment were measured. The drawn conclusion is that dqPCR performed using PanelChip™ is superior to the standard qPCR in terms of sensitivity, precision, and heparin tolerance. The dqPCR assay is a potential tool for clinical diagnosis and single-cell applications.

The single-species metagenome: subtyping Staphylococcus aureus core genome sequences from shotgun metagenomic data

PubMed Central

Li, Ben; Petit III, Robert A.; Qin, Zhaohui S.; Darrow, Lyndsey

2016-01-01

In this study we developed a genome-based method for detecting Staphylococcus aureus subtypes from metagenome shotgun sequence data. We used a binomial mixture model and the coverage counts at >100,000 known S. aureus SNP (single nucleotide polymorphism) sites derived from prior comparative genomic analysis to estimate the proportion of 40 subtypes in metagenome samples. We were able to obtain >87% sensitivity and >94% specificity at 0.025X coverage for S. aureus. We found that 321 and 149 metagenome samples from the Human Microbiome Project and metaSUB analysis of the New York City subway, respectively, contained S. aureus at genome coverage >0.025. In both projects, CC8 and CC30 were the most common S. aureus clonal complexes encountered. We found evidence that the subtype composition at different body sites of the same individual were more similar than random sampling and more limited evidence that certain body sites were enriched for particular subtypes. One surprising finding was the apparent high frequency of CC398, a lineage often associated with livestock, in samples from the tongue dorsum. Epidemiologic analysis of the HMP subject population suggested that high BMI (body mass index) and health insurance are possibly associated with S. aureus carriage but there was limited power to identify factors linked to carriage of even the most common subtype. In the NYC subway data, we found a small signal of geographic distance affecting subtype clustering but other unknown factors influence taxonomic distribution of the species around the city. PMID:27781166

Single Particle-Inductively Coupled Plasma Mass Spectroscopy Analysis of Metallic Nanoparticles in Environmental Samples with Large Dissolved Analyte Fractions.

PubMed

Schwertfeger, D M; Velicogna, Jessica R; Jesmer, Alexander H; Scroggins, Richard P; Princz, Juliska I

2016-10-18

There is an increasing interest to use single particle-inductively coupled plasma mass spectroscopy (SP-ICPMS) to help quantify exposure to engineered nanoparticles, and their transformation products, released into the environment. Hindering the use of this analytical technique for environmental samples is the presence of high levels of dissolved analyte which impedes resolution of the particle signal from the dissolved. While sample dilution is often necessary to achieve the low analyte concentrations necessary for SP-ICPMS analysis, and to reduce the occurrence of matrix effects on the analyte signal, it is used here to also reduce the dissolved signal relative to the particulate, while maintaining a matrix chemistry that promotes particle stability. We propose a simple, systematic dilution series approach where by the first dilution is used to quantify the dissolved analyte, the second is used to optimize the particle signal, and the third is used as an analytical quality control. Using simple suspensions of well characterized Au and Ag nanoparticles spiked with the dissolved analyte form, as well as suspensions of complex environmental media (i.e., extracts from soils previously contaminated with engineered silver nanoparticles), we show how this dilution series technique improves resolution of the particle signal which in turn improves the accuracy of particle counts, quantification of particulate mass and determination of particle size. The technique proposed here is meant to offer a systematic and reproducible approach to the SP-ICPMS analysis of environmental samples and improve the quality and consistency of data generated from this relatively new analytical tool.

Development of a sensitive and selective liquid chromatography-mass spectrometry method for high throughput analysis of paralytic shellfish toxins using graphitised carbon solid phase extraction.

PubMed

Boundy, Michael J; Selwood, Andrew I; Harwood, D Tim; McNabb, Paul S; Turner, Andrew D

2015-03-27

Routine regulatory monitoring of paralytic shellfish toxins (PST) commonly employs oxidative derivitisation and complex liquid chromatography fluorescence detection methods (LC-FL). The pre-column oxidation LC-FL method is currently implemented in New Zealand and the United Kingdom. When using this method positive samples are fractionated and two different oxidations are required to confirm the identity and quantity of each PST analogue present. There is a need for alternative methods that are simpler, provide faster turnaround times and have improved detection limits. Hydrophilic interaction liquid chromatography (HILIC) HPLC-MS/MS analysis of PST has been used for research purposes, but high detection limits and substantial sample matrix issues have prevented it from becoming a viable alternative for routine monitoring purposes. We have developed a HILIC UPLC-MS/MS method for paralytic shellfish toxins with an optimised desalting clean-up procedure on inexpensive carbon solid phase extraction cartridges for reduction of matrix interferences. This represents a major technical breakthrough and allows sensitive, selective and rapid analysis of paralytic shellfish toxins from a variety of sample types, including many commercially produced bivalve molluscan shellfish species. Additionally, this analytical approach avoids the need for complex calculations to determine sample toxicity, as unlike other methods each PST analogue is able to be quantified as a single resolved peak. This article presents the method development and optimisation information. A thorough single laboratory validation study has subsequently been performed and this data will be presented elsewhere. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of GHB in human hair by HPLC-MS/MS: Development and validation of a method and application to a study group and three possible single exposure cases.

PubMed

Bertol, Elisabetta; Mari, Francesco; Vaiano, Fabio; Romano, Guido; Zaami, Simona; Baglìo, Giovanni; Busardò, Francesco Paolo

2015-05-01

Gamma-hydroxybutyrate (GHB) over the last two decades has generated increased notoriety as a euphoric and disinhibiting drug of abuse in cases of drug-related sexual assault and for this reason it is considered a 'date rape' drug. The first aim of this paper was to develop and fully validate a method for the detection of GHB in human hair by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after liquid-liquid extraction (LLE). The second aim was the application of the method to hair samples of 30 GHB-free users in order to determine the basal level. The results obtained showed no significant differences in endogenous concentrations (p = 0.556) between hair samples of the three groups (black, blonde, and dyed hair) and the age and sex of the subjects did not affect the endogenous levels. Another 12 healthy volunteers, with no previous history of GHB use, were selected and a single dose (25 mg/Kg) was orally administered to all of them and hair samples were collected before the administration of the single dose and other two samples were collected one month and two months later, respectively. The segmental analysis of the latter two samples allowed us to calculate two ratios: 4.45:1 (95% C.I. 3.52-5.63) and 3.35:1 (95% C.I. 2.14-5.18), respectively, which can be recommended as reasonable values for a positive identification of GHB intake. Finally the method was applied to three real cases where a GHB single exposure probably occurred. Copyright © 2014 John Wiley & Sons, Ltd.

Fast Raman single bacteria identification: toward a routine in-vitro diagnostic

NASA Astrophysics Data System (ADS)

Douet, Alice; Josso, Quentin; Marchant, Adrien; Dutertre, Bertrand; Filiputti, Delphine; Novelli-Rousseau, Armelle; Espagnon, Isabelle; Kloster-Landsberg, Meike; Mallard, Frédéric; Perraut, Francois

2016-04-01

Timely microbiological results are essential to allow clinicians to optimize the prescribed treatment, ideally at the initial stage of the therapeutic process. Several approaches have been proposed to solve this issue and to provide the microbiological result in a few hours directly from the sample such as molecular biology. However fast and sensitive those methods are not based on single phenotypic information which presents several drawbacks and limitations. Optical methods have the advantage to allow single-cell sensitivity and to probe the phenotype of measured cells. Here we present a process and a prototype that allow automated single-bacteria phenotypic analysis. This prototype is based on the use of Digital In-line Holography techniques combined with a specially designed Raman spectrometer using a dedicated device to capture bacteria. The localization of single-cell is finely determined by using holograms and a proper propagation kernel. Holographic images are also used to analyze bacteria in the sample to sort potential pathogens from flora dwelling species or other biological particles. This accurate localization enables the use of a small confocal volume adapted to the measurement of single-cell. Along with the confocal volume adaptation, we also have modified every components of the spectrometer to optimize single-bacteria Raman measurements. This optimization allowed us to acquire informative single-cell spectra using an integration time of 0.5s only. Identification results obtained with this prototype are presented based on a 65144 Raman spectra database acquired automatically on 48 bacteria strains belonging to 8 species.

Parallel processing of genomics data

NASA Astrophysics Data System (ADS)

Agapito, Giuseppe; Guzzi, Pietro Hiram; Cannataro, Mario

2016-10-01

The availability of high-throughput experimental platforms for the analysis of biological samples, such as mass spectrometry, microarrays and Next Generation Sequencing, have made possible to analyze a whole genome in a single experiment. Such platforms produce an enormous volume of data per single experiment, thus the analysis of this enormous flow of data poses several challenges in term of data storage, preprocessing, and analysis. To face those issues, efficient, possibly parallel, bioinformatics software needs to be used to preprocess and analyze data, for instance to highlight genetic variation associated with complex diseases. In this paper we present a parallel algorithm for the parallel preprocessing and statistical analysis of genomics data, able to face high dimension of data and resulting in good response time. The proposed system is able to find statistically significant biological markers able to discriminate classes of patients that respond to drugs in different ways. Experiments performed on real and synthetic genomic datasets show good speed-up and scalability.

Preparative separation of underivatized amino acids for compound-specific stable isotope analysis and radiocarbon dating of hydrolyzed bone collagen.

PubMed

Tripp, Jennifer A; McCullagh, James S O; Hedges, Robert E M

2006-01-01

Analysis of stable and radioactive isotopes from bone collagen provides useful information to archaeologists about the origin and age of bone artifacts. Isolation and analysis of single amino acids from the proteins can provide additional and more accurate information by removing contamination and separating a bulk isotope signal into its constituent parts. In this paper, we report a new method for the separation and isolation of underivatized amino acids from bone collagen, and their analysis by isotope ratio MS and accelerator MS. RP chromatography is used to separate the amino acids with nonpolar side chains, followed by an ion pair separation to isolate the remaining amino acids. The method produces single amino acids with little or no contamination from the separation process and allows for the measurement of accurate stable isotope ratios and pure samples for radiocarbon dating.

SlideJ: An ImageJ plugin for automated processing of whole slide images.

PubMed

Della Mea, Vincenzo; Baroni, Giulia L; Pilutti, David; Di Loreto, Carla

2017-01-01

The digital slide, or Whole Slide Image, is a digital image, acquired with specific scanners, that represents a complete tissue sample or cytological specimen at microscopic level. While Whole Slide image analysis is recognized among the most interesting opportunities, the typical size of such images-up to Gpixels- can be very demanding in terms of memory requirements. Thus, while algorithms and tools for processing and analysis of single microscopic field images are available, Whole Slide images size makes the direct use of such tools prohibitive or impossible. In this work a plugin for ImageJ, named SlideJ, is proposed with the objective to seamlessly extend the application of image analysis algorithms implemented in ImageJ for single microscopic field images to a whole digital slide analysis. The plugin has been complemented by examples of macro in the ImageJ scripting language to demonstrate its use in concrete situations.

SlideJ: An ImageJ plugin for automated processing of whole slide images

PubMed Central

Baroni, Giulia L.; Pilutti, David; Di Loreto, Carla

2017-01-01

The digital slide, or Whole Slide Image, is a digital image, acquired with specific scanners, that represents a complete tissue sample or cytological specimen at microscopic level. While Whole Slide image analysis is recognized among the most interesting opportunities, the typical size of such images—up to Gpixels- can be very demanding in terms of memory requirements. Thus, while algorithms and tools for processing and analysis of single microscopic field images are available, Whole Slide images size makes the direct use of such tools prohibitive or impossible. In this work a plugin for ImageJ, named SlideJ, is proposed with the objective to seamlessly extend the application of image analysis algorithms implemented in ImageJ for single microscopic field images to a whole digital slide analysis. The plugin has been complemented by examples of macro in the ImageJ scripting language to demonstrate its use in concrete situations. PMID:28683129

Oufti: An integrated software package for high-accuracy, high-throughput quantitative microscopy analysis

PubMed Central

Paintdakhi, Ahmad; Parry, Bradley; Campos, Manuel; Irnov, Irnov; Elf, Johan; Surovtsev, Ivan; Jacobs-Wagner, Christine

2016-01-01

Summary With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today’s single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals, and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis, and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills. PMID:26538279

Principal component analysis and analysis of variance on the effects of Entellan New on the Raman spectra of fibers.

PubMed

Yu, Marcia M L; Sandercock, P Mark L

2012-01-01

During the forensic examination of textile fibers, fibers are usually mounted on glass slides for visual inspection and identification under the microscope. One method that has the capability to accurately identify single textile fibers without subsequent demounting is Raman microspectroscopy. The effect of the mountant Entellan New on the Raman spectra of fibers was investigated to determine if it is suitable for fiber analysis. Raman spectra of synthetic fibers mounted in three different ways were collected and subjected to multivariate analysis. Principal component analysis score plots revealed that while spectra from different fiber classes formed distinct groups, fibers of the same class formed a single group regardless of the mounting method. The spectra of bare fibers and those mounted in Entellan New were found to be statistically indistinguishable by analysis of variance calculations. These results demonstrate that fibers mounted in Entellan New may be identified directly by Raman microspectroscopy without further sample preparation. © 2011 American Academy of Forensic Sciences.

Development of a sensitivity analysis technique for multiloop flight control systems

NASA Technical Reports Server (NTRS)

Vaillard, A. H.; Paduano, J.; Downing, D. R.

1985-01-01

This report presents the development and application of a sensitivity analysis technique for multiloop flight control systems. This analysis yields very useful information on the sensitivity of the relative-stability criteria of the control system, with variations or uncertainties in the system and controller elements. The sensitivity analysis technique developed is based on the computation of the singular values and singular-value gradients of a feedback-control system. The method is applicable to single-input/single-output as well as multiloop continuous-control systems. Application to sampled-data systems is also explored. The sensitivity analysis technique was applied to a continuous yaw/roll damper stability augmentation system of a typical business jet, and the results show that the analysis is very useful in determining the system elements which have the largest effect on the relative stability of the closed-loop system. As a secondary product of the research reported here, the relative stability criteria based on the concept of singular values were explored.

Identification of mineral compositions in some renal calculi by FT Raman and IR spectral analysis

NASA Astrophysics Data System (ADS)

Tonannavar, J.; Deshpande, Gouri; Yenagi, Jayashree; Patil, Siddanagouda B.; Patil, Nikhil A.; Mulimani, B. G.

2016-02-01

We present in this paper accurate and reliable Raman and IR spectral identification of mineral constituents in nine samples of renal calculi (kidney stones) removed from patients suffering from nephrolithiasis. The identified mineral components include Calcium Oxalate Monohydrate (COM, whewellite), Calcium Oxalate Dihydrate (COD, weddellite), Magnesium Ammonium Phosphate Hexahydrate (MAPH, struvite), Calcium Hydrogen Phosphate Dihydrate (CHPD, brushite), Pentacalcium Hydroxy Triphosphate (PCHT, hydroxyapatite) and Uric Acid (UA). The identification is based on a satisfactory assignment of all the observed IR and Raman bands (3500-400 cm- 1) to chemical functional groups of mineral components in the samples, aided by spectral analysis of pure materials of COM, MAPH, CHPD and UA. It is found that the eight samples are composed of COM as the common component, the other mineral species as common components are: MAPH in five samples, PCHT in three samples, COD in three samples, UA in three samples and CHPD in two samples. One sample is wholly composed of UA as a single component; this inference is supported by the good agreement between ab initio density functional theoretical spectra and experimental spectral measurements of both sample and pure material. A combined application of Raman and IR techniques has shown that, where the IR is ambiguous, the Raman analysis can differentiate COD from COM and PCHT from MAPH.

Identification of mineral compositions in some renal calculi by FT Raman and IR spectral analysis.

PubMed

Tonannavar, J; Deshpande, Gouri; Yenagi, Jayashree; Patil, Siddanagouda B; Patil, Nikhil A; Mulimani, B G

2016-02-05

We present in this paper accurate and reliable Raman and IR spectral identification of mineral constituents in nine samples of renal calculi (kidney stones) removed from patients suffering from nephrolithiasis. The identified mineral components include Calcium Oxalate Monohydrate (COM, whewellite), Calcium Oxalate Dihydrate (COD, weddellite), Magnesium Ammonium Phosphate Hexahydrate (MAPH, struvite), Calcium Hydrogen Phosphate Dihydrate (CHPD, brushite), Pentacalcium Hydroxy Triphosphate (PCHT, hydroxyapatite) and Uric Acid (UA). The identification is based on a satisfactory assignment of all the observed IR and Raman bands (3500-400c m(-1)) to chemical functional groups of mineral components in the samples, aided by spectral analysis of pure materials of COM, MAPH, CHPD and UA. It is found that the eight samples are composed of COM as the common component, the other mineral species as common components are: MAPH in five samples, PCHT in three samples, COD in three samples, UA in three samples and CHPD in two samples. One sample is wholly composed of UA as a single component; this inference is supported by the good agreement between ab initio density functional theoretical spectra and experimental spectral measurements of both sample and pure material. A combined application of Raman and IR techniques has shown that, where the IR is ambiguous, the Raman analysis can differentiate COD from COM and PCHT from MAPH. Copyright © 2015 Elsevier B.V. All rights reserved.

Single exosome detection in serum using microtoroid optical resonators (Conference Presentation)

NASA Astrophysics Data System (ADS)

Su, Judith

2016-03-01

Recently exosomes have attracted interest due to their potential as cancer biomarkers. We report the real time, label-free sensing of single exosomes in serum using microtoroid optical resonators. We use this approach to assay the progression of tumors implanted in mice by specifically detecting low concentrations of tumor-derived exosomes. Our approach measures the adsorption of individual exosomes onto a functionalized silica microtoroid by tracking changes in the optical resonant frequency of the microtoroid. When exosomes land on the microtoroid, they perturb its refractive index in the evanescent field and thus shift its resonance frequency. Through digital frequency locking, we are able to rapidly track these shifts with accuracies of better than 10 attometers (one part in 10^11). Samples taken from tumor-implanted mice from later weeks generated larger frequency shifts than those from earlier weeks. Control samples taken from a mouse with no tumor generated no such increase in signal between subsequent weeks. Analysis of shifts from tumor-implanted mouse samples show a distribution of unitary steps, with the maximum step having a height of ~1.2 fm, corresponding to an exosome size of 44 ± 4.8 nm. This size range corresponds to that found by performing nanoparticle tracking analysis on the same samples. Our results demonstrate development towards a minimally-invasive tumor "biopsy" that eliminates the need to find and access a tumor.

Discrimination of Oil Slicks and Lookalikes in Polarimetric SAR Images Using CNN.

PubMed

Guo, Hao; Wu, Danni; An, Jubai

2017-08-09

Oil slicks and lookalikes (e.g., plant oil and oil emulsion) all appear as dark areas in polarimetric Synthetic Aperture Radar (SAR) images and are highly heterogeneous, so it is very difficult to use a single feature that can allow classification of dark objects in polarimetric SAR images as oil slicks or lookalikes. We established multi-feature fusion to support the discrimination of oil slicks and lookalikes. In the paper, simple discrimination analysis is used to rationalize a preferred features subset. The features analyzed include entropy, alpha, and Single-bounce Eigenvalue Relative Difference (SERD) in the C-band polarimetric mode. We also propose a novel SAR image discrimination method for oil slicks and lookalikes based on Convolutional Neural Network (CNN). The regions of interest are selected as the training and testing samples for CNN on the three kinds of polarimetric feature images. The proposed method is applied to a training data set of 5400 samples, including 1800 crude oil, 1800 plant oil, and 1800 oil emulsion samples. In the end, the effectiveness of the method is demonstrated through the analysis of some experimental results. The classification accuracy obtained using 900 samples of test data is 91.33%. It is here observed that the proposed method not only can accurately identify the dark spots on SAR images but also verify the ability of the proposed algorithm to classify unstructured features.

Discrimination of Oil Slicks and Lookalikes in Polarimetric SAR Images Using CNN

PubMed Central

An, Jubai

2017-01-01

Oil slicks and lookalikes (e.g., plant oil and oil emulsion) all appear as dark areas in polarimetric Synthetic Aperture Radar (SAR) images and are highly heterogeneous, so it is very difficult to use a single feature that can allow classification of dark objects in polarimetric SAR images as oil slicks or lookalikes. We established multi-feature fusion to support the discrimination of oil slicks and lookalikes. In the paper, simple discrimination analysis is used to rationalize a preferred features subset. The features analyzed include entropy, alpha, and Single-bounce Eigenvalue Relative Difference (SERD) in the C-band polarimetric mode. We also propose a novel SAR image discrimination method for oil slicks and lookalikes based on Convolutional Neural Network (CNN). The regions of interest are selected as the training and testing samples for CNN on the three kinds of polarimetric feature images. The proposed method is applied to a training data set of 5400 samples, including 1800 crude oil, 1800 plant oil, and 1800 oil emulsion samples. In the end, the effectiveness of the method is demonstrated through the analysis of some experimental results. The classification accuracy obtained using 900 samples of test data is 91.33%. It is here observed that the proposed method not only can accurately identify the dark spots on SAR images but also verify the ability of the proposed algorithm to classify unstructured features. PMID:28792477

Effect of sampling and diagnostic effort on the assessment of schistosomiasis and soil-transmitted helminthiasis and drug efficacy: a meta-analysis of six drug efficacy trials and one epidemiological survey.

PubMed

Levecke, Bruno; Brooker, Simon J; Knopp, Stefanie; Steinmann, Peter; Sousa-Figueiredo, Jose Carlos; Stothard, J Russell; Utzinger, Jürg; Vercruysse, Jozef

2014-12-01

It is generally recommended to perform multiple stool examinations in order to improve the diagnostic accuracy when assessing the impact of mass drug administration programmes to control human intestinal worm infections and determining efficacy of the drugs administered. However, the collection and diagnostic work-up of multiple stool samples increases costs and workload. It has been hypothesized that these increased efforts provide more accurate results when infection and drug efficacy are summarized by prevalence (proportion of subjects infected) and cure rate (CR, proportion of infected subjects that become egg-negative after drug administration), respectively, but not when these indicators are expressed in terms of infection intensity and egg reduction rate (ERR). We performed a meta-analysis of six drug efficacy trials and one epidemiological survey. We compared prevalence and intensity of infection, CR and ERR based on collection of one or two stool samples that were processed with single or duplicate Kato-Katz thick smears. We found that the accuracy of prevalence estimates and CR was lowest with the minimal sampling effort, but that this was not the case for estimating infection intensity and ERR. Hence, a single Kato-Katz thick smear is sufficient for reporting infection intensity and ERR following drug treatment.

Preparation of longitudinal sections of hair samples for the analysis of cocaine by MALDI-MS/MS and TOF-SIMS imaging.

PubMed

Flinders, Bryn; Cuypers, Eva; Zeijlemaker, Hans; Tytgat, Jan; Heeren, Ron M A

2015-10-01

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) for the analysis of intact hair is a powerful tool for the detection of drugs of abuse in toxicology and forensic applications. Here we present a quick, easy, and reproducible method of preparing longitudinal sections of single hairs. This method improves the accessibility of chemicals embedded in the hair matrix for molecular imaging with mass spectrometry. The images obtained from a single, sectioned hair sample show molecular distributions in the exposed medulla, cortex, and a portion of the cuticle observed as a narrow layer surrounding the cortex. Using MALDI-MS/MS imaging, the distribution of cocaine was observed throughout five longitudinally sectioned drug-user hair samples. The images showed the distribution of the product ion at m/z 182, derived from the precursor ion of cocaine at m/z 304. MetA-SIMS images of longitudinally sectioned hair samples showed a more detailed distribution of cocaine at m/z 304, benzoylecgonine the major metabolite of cocaine at m/z 290 and other drugs such as methadone which was observed at m/z 310. Chronological information of drug intake can be obtained more sensitively. The chronological detail is in hours rather than months, which is of great interest in clinical as well as forensic applications. Copyright © 2015 John Wiley & Sons, Ltd.

Hyperspectral techniques in analysis of oral dosage forms.

PubMed

Hamilton, Sara J; Lowell, Amanda E; Lodder, Robert A

2002-10-01

Pharmaceutical oral dosage forms are used in this paper to test the sensitivity and spatial resolution of hyperspectral imaging instruments. The first experiment tested the hypothesis that a near-infrared (IR) tunable diode-based remote sensing system is capable of monitoring degradation of hard gelatin capsules at a relatively long distance (0.5 km). Spectra from the capsules were used to differentiate among capsules exposed to an atmosphere containing 150 ppb formaldehyde for 0, 2, 4, and 8 h. Robust median-based principal component regression with Bayesian inference was employed for outlier detection. The second experiment tested the hypothesis that near-IR imaging spectrometry of tablets permits the identification and composition of multiple individual tablets to be determined simultaneously. A near-IR camera was used to collect thousands of spectra simultaneously from a field of blister-packaged tablets. The number of tablets that a typical near-IR camera can currently analyze simultaneously was estimated to be approximately 1300. The bootstrap error-adjusted single-sample technique chemometric-imaging algorithm was used to draw probability-density contour plots that revealed tablet composition. The single-capsule analysis provides an indication of how far apart the sample and instrumentation can be and still maintain adequate signal-to-noise ratio (S/N), while the multiple-tablet imaging experiment gives an indication of how many samples can be analyzed simultaneously while maintaining an adequate S/N and pixel coverage on each sample.

A novel quantitation approach for maximizing detectable targets for offensive/volatile odorants with diverse functional groups by thermal desorption-gas chromatography-mass spectrometry

NASA Astrophysics Data System (ADS)

Kim, Yong-Hyun; Kim, Ki-Hyun

2016-07-01

A multitude of analytical systems are needed to analyze diverse odorants with various functionalities. In this study, an experimental method was developed to assess the maximum covering range of odorants using a single experimental setup consisting of a thermal desorber-gas chromatography-mass spectrometry system. To this end, a total of 20 offensive odorants (aldehyde, ketone, ester, alcohol, aromatic, sulfide, amine, and carboxyl) were selected and tested by a single system. The analytical results of standards and environmental samples were evaluated in a number of respects. In the analysis of the standards, all targets were quantified via Carbopack (C + B + X) tube sampling while operating the thermal desorber at -25 °C. The method detection limits of 18 targets (exception of 2 out of the 20 targets: acetaldehyde and methanethiol) were excellent (mean 0.04 ± 0.03 ppb) in terms of their odor threshold values (74.7 ± 140 ~ 624 ± 1,729 ppb). The analysis of organic fertilizer plant samples at a pig farm (slurry treatment facility, compost facility, and ambient air) confirmed the presence of 18 odorants from 0.03 ppb (dimethyldisulfide, ambient sample) to 522 ppb (methyl ethyl ketone, slurry treatment facility). As such, our method allowed simultaneous quantitation of most key odorants with sufficient reliability and sensitivity.

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

NASA Astrophysics Data System (ADS)

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-04-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

Single-molecule analysis of steroid receptor and cofactor action in living cells

PubMed Central

Paakinaho, Ville; Presman, Diego M.; Ball, David A.; Johnson, Thomas A.; Schiltz, R. Louis; Levitt, Peter; Mazza, Davide; Morisaki, Tatsuya; Karpova, Tatiana S.; Hager, Gordon L.

2017-01-01

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other’s dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level. PMID:28635963

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

PubMed Central

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-01-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue. PMID:27063397

Comprehensive comparative analysis of 5'-end RNA-sequencing methods.

PubMed

Adiconis, Xian; Haber, Adam L; Simmons, Sean K; Levy Moonshine, Ami; Ji, Zhe; Busby, Michele A; Shi, Xi; Jacques, Justin; Lancaster, Madeline A; Pan, Jen Q; Regev, Aviv; Levin, Joshua Z

2018-06-04

Specialized RNA-seq methods are required to identify the 5' ends of transcripts, which are critical for studies of gene regulation, but these methods have not been systematically benchmarked. We directly compared six such methods, including the performance of five methods on a single human cellular RNA sample and a new spike-in RNA assay that helps circumvent challenges resulting from uncertainties in annotation and RNA processing. We found that the 'cap analysis of gene expression' (CAGE) method performed best for mRNA and that most of its unannotated peaks were supported by evidence from other genomic methods. We applied CAGE to eight brain-related samples and determined sample-specific transcription start site (TSS) usage, as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.

Rapid screening of illicit additives in weight loss dietary supplements with desorption corona beam ionisation (DCBI) mass spectrometry.

PubMed

Wang, H; Wu, Y; Zhao, Y; Sun, W; Ding, L; Guo, B; Chen, B

2012-08-01

Desorption corona beam ionisation (DCBI), the relatively novel ambient mass spectrometry (MS) technique, was utilised to screen for illicit additives in weight-loss food. The five usually abused chemicals - fenfluramine, N-di-desmethyl sibutramine, N-mono-desmethyl sibutramine, sibutramine and phenolphthalein - were detected with the proposed DCBI-MS method. Fast single-sample and high-throughput analysis was demonstrated. Semi-quantification was accomplished based on peak areas in the ion chromatograms. Four illicit additives were identified and semi-quantified in commercial samples. As there was no tedious sample pre-treatment compared with conventional HPLC methods, high-throughput analysis was achieved with DCBI. The results proved that DCBI-MS is a powerful tool for the rapid screening of illicit additives in weight-loss dietary supplements.

Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process.

PubMed

Wen, Xianyu; Jeong, Seorin; Kim, Younghoon; Bae, Jeong Mo; Cho, Nam Yun; Kim, Jung Ho; Kang, Gyeong Hoon

2017-01-01

Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively. Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 °C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer. Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.

Adolescents' multiple versus single primary attachment figures, reorganization of attachment hierarchy, and adjustments: the important people interview approach.

PubMed

Umemura, Tomotaka; Lacinová, Lenka; Kraus, Jakub; Horská, Eliška; Pivodová, Lenka

2018-04-20

Using 212 adolescents from a central-European country (mean age = 14.02, SD = 2.05, ranged from 11 to 18 years; females = 54%) and a multi-informant method to measure adolescents' behavioral and emotional adjustments, the present study explored three aspects regarding the attachment hierarchy. (1) The three types of behavioral systems of Rosenthal and Kobak's important people interview (IPI) were initially validated using an exploratory factor analysis with a US sample. Using a confirmatory factor analysis with a Czech sample, we replicated these three behavioral systems: attachment bond, support seeking, and affiliation. (2) We found that adolescents who developed attachment bond to multiple primary attachment figures were likely to score lower on both teacher-rated and parent-rated internalizing problems compared to those who had a single primary attachment figure. These multiple primary attachment figures tended to be family members (not peers). (3) Early adolescents who placed parents low in their attachment hierarchy scored higher on self-reported negative affect and lower on self-reported positive affect compared to early adolescents who placed parents high. The present study highlights multiple (vs. single) primary attachment figures as a protective factor and the premature reorganization of attachment hierarchy as a risk factor for adolescents' emotional and affective adjustments.

Comprehensive genetic testing for female and male infertility using next-generation sequencing.

PubMed

Patel, Bonny; Parets, Sasha; Akana, Matthew; Kellogg, Gregory; Jansen, Michael; Chang, Chihyu; Cai, Ying; Fox, Rebecca; Niknazar, Mohammad; Shraga, Roman; Hunter, Colby; Pollock, Andrew; Wisotzkey, Robert; Jaremko, Malgorzata; Bisignano, Alex; Puig, Oscar

2018-05-19

To develop a comprehensive genetic test for female and male infertility in support of medical decisions during assisted reproductive technology (ART) protocols. We developed a next-generation sequencing (NGS) gene panel consisting of 87 genes including promoters, 5' and 3' untranslated regions, exons, and selected introns. In addition, sex chromosome aneuploidies and Y chromosome microdeletions were analyzed concomitantly using the same panel. The NGS panel was analytically validated by retrospective analysis of 118 genomic DNA samples with known variants in loci representative of female and male infertility. Our results showed analytical accuracy of > 99%, with > 98% sensitivity for single-nucleotide variants (SNVs) and > 91% sensitivity for insertions/deletions (indels). Clinical sensitivity was assessed with samples containing variants representative of male and female infertility, and it was 100% for SNVs/indels, CFTR IVS8-5T variants, sex chromosome aneuploidies, and copy number variants (CNVs) and > 93% for Y chromosome microdeletions. Cost analysis shows potential savings when comparing this single NGS assay with the standard approach, which includes multiple assays. A single, comprehensive, NGS panel can simplify the ordering process for healthcare providers, reduce turnaround time, and lower the overall cost of testing for genetic assessment of infertility in females and males, while maintaining accuracy.

Microfluidic cell isolation technology for drug testing of single tumor cells and their clusters.

PubMed

Bithi, Swastika S; Vanapalli, Siva A

2017-02-02

Drug assays with patient-derived cells such as circulating tumor cells requires manipulating small sample volumes without loss of rare disease-causing cells. Here, we report an effective technology for isolating and analyzing individual tumor cells and their clusters from minute sample volumes using an optimized microfluidic device integrated with pipettes. The method involves using hand pipetting to create an array of cell-laden nanoliter-sized droplets immobilized in a microfluidic device without loss of tumor cells during the pipetting process. Using this technology, we demonstrate single-cell analysis of tumor cell response to the chemotherapy drug doxorubicin. We find that even though individual tumor cells display diverse uptake profiles of the drug, the onset of apoptosis is determined by accumulation of a critical intracellular concentration of doxorubicin. Experiments with clusters of tumor cells compartmentalized in microfluidic drops reveal that cells within a cluster have higher viability than their single-cell counterparts when exposed to doxorubicin. This result suggests that circulating tumor cell clusters might be able to better survive chemotherapy drug treatment. Our technology is a promising tool for understanding tumor cell-drug interactions in patient-derived samples including rare cells.

Precise turnaround time measurement of laboratory processes using radiofrequency identification technology.

PubMed

Mayer, Horst; Brümmer, Jens; Brinkmann, Thomas

2011-01-01

To implement Lean Six Sigma in our central laboratory we conducted a project to measure single pre-analytical steps influencing turnaround time (TAT) of emergency department (ED) serum samples. The traditional approach of extracting data from the Laboratory Information System (LIS) for a retrospective calculation of a mean TAT is not suitable. Therefore, we used radiofrequency identification (RFID) chips for real time tracking of individual samples at any pre-analytical step. 1,200 serum tubes were labelled with RFID chips and were provided to the emergency department. 3 RFID receivers were installed in the laboratory: at the outlet of the pneumatic tube system, at the centrifuge, and in the analyser area. In addition, time stamps of sample entry at the automated sample distributor and communication of results from the analyser were collected from LIS. 1,023 labelled serum tubes arrived at our laboratory. 899 RFID tags were used for TAT calculation. The following transfer times were determined (median 95th percentile in min:sec): pneumatic tube system --> centrifuge (01:25/04:48), centrifuge --> sample distributor (14:06/5:33), sample distributor --> analysis system zone (02:39/15:07), analysis system zone --> result communication (12:42/22:21). Total TAT was calculated at 33:19/57:40 min:sec. Manual processes around centrifugation were identified as a major part of TAT with 44%/60% (median/95th percentile). RFID is a robust, easy to use, and error-free technology and not susceptible to interferences in the laboratory environment. With this study design we were able to measure significant variations in a single manual sample transfer process. We showed that TAT is mainly influenced by manual steps around the centrifugation process and we concluded that centrifugation should be integrated in solutions for total laboratory automation.

Correcting for intra-experiment variation in Illumina BeadChip data is necessary to generate robust gene-expression profiles.

PubMed

Kitchen, Robert R; Sabine, Vicky S; Sims, Andrew H; Macaskill, E Jane; Renshaw, Lorna; Thomas, Jeremy S; van Hemert, Jano I; Dixon, J Michael; Bartlett, John M S

2010-02-24

Microarray technology is a popular means of producing whole genome transcriptional profiles, however high cost and scarcity of mRNA has led many studies to be conducted based on the analysis of single samples. We exploit the design of the Illumina platform, specifically multiple arrays on each chip, to evaluate intra-experiment technical variation using repeated hybridisations of universal human reference RNA (UHRR) and duplicate hybridisations of primary breast tumour samples from a clinical study. A clear batch-specific bias was detected in the measured expressions of both the UHRR and clinical samples. This bias was found to persist following standard microarray normalisation techniques. However, when mean-centering or empirical Bayes batch-correction methods (ComBat) were applied to the data, inter-batch variation in the UHRR and clinical samples were greatly reduced. Correlation between replicate UHRR samples improved by two orders of magnitude following batch-correction using ComBat (ranging from 0.9833-0.9991 to 0.9997-0.9999) and increased the consistency of the gene-lists from the duplicate clinical samples, from 11.6% in quantile normalised data to 66.4% in batch-corrected data. The use of UHRR as an inter-batch calibrator provided a small additional benefit when used in conjunction with ComBat, further increasing the agreement between the two gene-lists, up to 74.1%. In the interests of practicalities and cost, these results suggest that single samples can generate reliable data, but only after careful compensation for technical bias in the experiment. We recommend that investigators appreciate the propensity for such variation in the design stages of a microarray experiment and that the use of suitable correction methods become routine during the statistical analysis of the data.

Correcting for intra-experiment variation in Illumina BeadChip data is necessary to generate robust gene-expression profiles

PubMed Central

2010-01-01

Background Microarray technology is a popular means of producing whole genome transcriptional profiles, however high cost and scarcity of mRNA has led many studies to be conducted based on the analysis of single samples. We exploit the design of the Illumina platform, specifically multiple arrays on each chip, to evaluate intra-experiment technical variation using repeated hybridisations of universal human reference RNA (UHRR) and duplicate hybridisations of primary breast tumour samples from a clinical study. Results A clear batch-specific bias was detected in the measured expressions of both the UHRR and clinical samples. This bias was found to persist following standard microarray normalisation techniques. However, when mean-centering or empirical Bayes batch-correction methods (ComBat) were applied to the data, inter-batch variation in the UHRR and clinical samples were greatly reduced. Correlation between replicate UHRR samples improved by two orders of magnitude following batch-correction using ComBat (ranging from 0.9833-0.9991 to 0.9997-0.9999) and increased the consistency of the gene-lists from the duplicate clinical samples, from 11.6% in quantile normalised data to 66.4% in batch-corrected data. The use of UHRR as an inter-batch calibrator provided a small additional benefit when used in conjunction with ComBat, further increasing the agreement between the two gene-lists, up to 74.1%. Conclusion In the interests of practicalities and cost, these results suggest that single samples can generate reliable data, but only after careful compensation for technical bias in the experiment. We recommend that investigators appreciate the propensity for such variation in the design stages of a microarray experiment and that the use of suitable correction methods become routine during the statistical analysis of the data. PMID:20181233

Individual detection of genetically modified maize varieties in non-identity-preserved maize samples.

PubMed

Akiyama, Hiroshi; Sakata, Kozue; Kondo, Kazunari; Tanaka, Asako; Liu, Ming S; Oguchi, Taichi; Furui, Satoshi; Kitta, Kazumi; Hino, Akihiro; Teshima, Reiko

2008-03-26

In many countries, the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved GM varieties. The GMO content in a maize sample containing the combined-trait (stacked) GM maize as determined by the currently available methodology is likely to be overestimated. However, there has been little information in the literature on the mixing level and varieties of stacked GM maize in real sample grains. For the first time, the GMO content of non-identity-preserved (non-IP) maize samples imported from the United States has been successfully determined by using a previously developed individual kernel detection system coupled to a multiplex qualitative PCR method followed by multichannel capillary gel electrophoresis system analysis. To clarify the GMO content in the maize samples imported from the United States, determine how many stacked GM traits are contained therein, and which GM trait varieties frequently appeared in 2005, the GMO content (percent) on a kernel basis and the varieties of the GM kernels in the non-IP maize samples imported from the United States were investigated using the individual kernel analysis system. The average (+/-standard deviation) of the GMO contents on a kernel basis in five non-IP sample lots was determined to be 51.0+/-21.6%, the percentage of a single GM trait grains was 39%, and the percentage of the stacked GM trait grains was 12%. The MON810 grains and NK603 grains were the most frequent varieties in the single GM traits. The most frequent stacked GM traits were the MON810xNK603 grains. In addition, the present study would provide the answer and impact for the quantification of GM maize content in the GM maize kernels on labeling regulation.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling

NASA Astrophysics Data System (ADS)

Comi, Troy J.; Neumann, Elizabeth K.; Do, Thanh D.; Sweedler, Jonathan V.

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. [Figure not available: see fulltext.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling.

PubMed

Comi, Troy J; Neumann, Elizabeth K; Do, Thanh D; Sweedler, Jonathan V

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. Graphical Abstract ᅟ.

Effects of sample treatments on genome recovery via single-cell genomics

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

Clingenpeel, Scott; Schwientek, Patrick; Hugenholtz, Philip

2014-06-13

It is known that single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we demonstrate that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis.

PubMed

Lefterova, Martina I; Shen, Peidong; Odegaard, Justin I; Fung, Eula; Chiang, Tsoyu; Peng, Gang; Davis, Ronald W; Wang, Wenyi; Kharrazi, Martin; Schrijver, Iris; Scharfe, Curt

2016-03-01

Newborn screening for cystic fibrosis enables early detection and management of this debilitating genetic disease. Implementing comprehensive CFTR analysis using Sanger sequencing as a component of confirmatory testing of all screen-positive newborns has remained impractical due to relatively lengthy turnaround times and high cost. Here, we describe CFseq, a highly sensitive, specific, rapid (<3 days), and cost-effective assay for comprehensive CFTR gene analysis from dried blood spots, the common newborn screening specimen. The unique design of CFseq integrates optimized dried blood spot sample processing, a novel multiplex amplification method from as little as 1 ng of genomic DNA, and multiplex next-generation sequencing of 96 samples in a single run to detect all relevant CFTR mutation types. Sequence data analysis utilizes publicly available software supplemented by an expert-curated compendium of >2000 CFTR variants. Validation studies across 190 dried blood spots demonstrated 100% sensitivity and a positive predictive value of 100% for single-nucleotide variants and insertions and deletions and complete concordance across the polymorphic poly-TG and consecutive poly-T tracts. Additionally, we accurately detected both a known exon 2,3 deletion and a previously undetected exon 22,23 deletion. CFseq is thus able to replace all existing CFTR molecular assays with a single robust, definitive assay at significant cost and time savings and could be adapted to high-throughput screening of other inherited conditions. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Redefining the WISC-R: Implications for Professional Practice and Public Policy.

ERIC Educational Resources Information Center

Macmann, Gregg M.; Barnett, David W.

1992-01-01

The factor structure of the Wechsler Intelligence Scale for Children (Revised) was examined in the standardization sample using new methods of factor analysis. The substantial overlap across factors was most parsimoniously represented by a single general factor. Implications for public policy regarding the purposes and outcomes of special…

Simultaneous analysis of fourteen endogenous steroid hormones by liquid chromatography tandem mass spectrometry with atmospheric pressure photoionization

EPA Science Inventory

Product Description: To understand how some chemicals affect the endocrine system, controlled lab experiments often monitor how chemicals impact natural steroid hormones in fish. Current methods can target only one or two hormones in a single sample, limiting the information that...

Integrated crystal mounting and alignment system for high-throughput biological crystallography

DOEpatents

Nordmeyer, Robert A.; Snell, Gyorgy P.; Cornell, Earl W.; Kolbe, William F.; Yegian, Derek T.; Earnest, Thomas N.; Jaklevich, Joseph M.; Cork, Carl W.; Santarsiero, Bernard D.; Stevens, Raymond C.

2007-09-25

A method and apparatus for the transportation, remote and unattended mounting, and visual alignment and monitoring of protein crystals for synchrotron generated x-ray diffraction analysis. The protein samples are maintained at liquid nitrogen temperatures at all times: during shipment, before mounting, mounting, alignment, data acquisition and following removal. The samples must additionally be stably aligned to within a few microns at a point in space. The ability to accurately perform these tasks remotely and automatically leads to a significant increase in sample throughput and reliability for high-volume protein characterization efforts. Since the protein samples are placed in a shipping-compatible layered stack of sample cassettes each holding many samples, a large number of samples can be shipped in a single cryogenic shipping container.

Integrated crystal mounting and alignment system for high-throughput biological crystallography

DOEpatents

Nordmeyer, Robert A.; Snell, Gyorgy P.; Cornell, Earl W.; Kolbe, William; Yegian, Derek; Earnest, Thomas N.; Jaklevic, Joseph M.; Cork, Carl W.; Santarsiero, Bernard D.; Stevens, Raymond C.

2005-07-19

A method and apparatus for the transportation, remote and unattended mounting, and visual alignment and monitoring of protein crystals for synchrotron generated x-ray diffraction analysis. The protein samples are maintained at liquid nitrogen temperatures at all times: during shipment, before mounting, mounting, alignment, data acquisition and following removal. The samples must additionally be stably aligned to within a few microns at a point in space. The ability to accurately perform these tasks remotely and automatically leads to a significant increase in sample throughput and reliability for high-volume protein characterization efforts. Since the protein samples are placed in a shipping-compatible layered stack of sample cassettes each holding many samples, a large number of samples can be shipped in a single cryogenic shipping container.

Distribution and Taxonomic Significance of Secondary Metabolites Occurring in the Methanol Extracts of the Stonecrops (Sedum L., Crassulaceae) from the Central Balkan Peninsula.

PubMed

Stojanovic, Gordana S; Jovanović, Snežana C; Zlatković, Bojan K

2015-06-01

The present study is engaged in the chemical composition of methanol extracts of Sedum taxa from the central part of the Balkan Peninsula, and representatives from other genera of Crassulaceae (Crassula, Echeveria and Kalanchoe) considered as out-groups. The chemical composition of extracts was determined by HPLC analysis, according to retention time of standards and characteristic absorption spectra of components. Identified components were considered as original variables with possible chemotaxonomic significance. Relationships of examined plant samples were investigated by agglomerative hierarchical cluster analysis (AHC). The obtained results showed how the distribution of methanol extract components (mostly phenolics) affected grouping of the examined samples. The obtained clustering showed satisfactory grouping of the examined samples, among which some representatives of the Sedum series, Rupestria and Magellensia, are the most remote. The out-group samples were not clearly singled out with regard to Sedum samples as expected; this especially applies to samples of Crassula ovata and Echeveria lilacina, while Kalanchoe daigremontiana was more separated from most of the Sedum samples.

An integratable microfluidic cartridge for forensic swab samples lysis.

PubMed

Yang, Jianing; Brooks, Carla; Estes, Matthew D; Hurth, Cedric M; Zenhausern, Frederic

2014-01-01

Fully automated rapid forensic DNA analysis requires integrating several multistep processes onto a single microfluidic platform, including substrate lysis, extraction of DNA from the released lysate solution, multiplexed PCR amplification of STR loci, separation of PCR products by capillary electrophoresis, and analysis for allelic peak calling. Over the past several years, most of the rapid DNA analysis systems developed started with the reference swab sample lysate and involved an off-chip lysis of collected substrates. As a result of advancement in technology and chemistry, addition of a microfluidic module for swab sample lysis has been achieved in a few of the rapid DNA analysis systems. However, recent reports on integrated rapid DNA analysis systems with swab-in and answer-out capability lack any quantitative and qualitative characterization of the swab-in sample lysis module, which is important for downstream forensic sample processing. Maximal collection and subsequent recovery of the biological material from the crime scene is one of the first and critical steps in forensic DNA technology. Herein we present the design, fabrication and characterization of an integratable swab lysis cartridge module and the test results obtained from different types of commonly used forensic swab samples, including buccal, saliva, and blood swab samples, demonstrating the compatibility with different downstream DNA extraction chemistries. This swab lysis cartridge module is easy to operate, compatible with both forensic and microfluidic requirements, and ready to be integrated with our existing automated rapid forensic DNA analysis system. Following the characterization of the swab lysis module, an integrated run from buccal swab sample-in to the microchip CE electropherogram-out was demonstrated on the integrated prototype instrument. Therefore, in this study, we demonstrate that this swab lysis cartridge module is: (1) functionally, comparable with routine benchtop lysis, (2) compatible with various types of swab samples and chemistries, and (3) integratable to achieve a micro total analysis system (μTAS) for rapid DNA analysis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

High Sensitivity and High Detection Specificity of Gold-Nanoparticle-Grafted Nanostructured Silicon Mass Spectrometry for Glucose Analysis.

PubMed

Tsao, Chia-Wen; Yang, Zhi-Jie

2015-10-14

Desorption/ionization on silicon (DIOS) is a high-performance matrix-free mass spectrometry (MS) analysis method that involves using silicon nanostructures as a matrix for MS desorption/ionization. In this study, gold nanoparticles grafted onto a nanostructured silicon (AuNPs-nSi) surface were demonstrated as a DIOS-MS analysis approach with high sensitivity and high detection specificity for glucose detection. A glucose sample deposited on the AuNPs-nSi surface was directly catalyzed to negatively charged gluconic acid molecules on a single AuNPs-nSi chip for MS analysis. The AuNPs-nSi surface was fabricated using two electroless deposition steps and one electroless etching step. The effects of the electroless fabrication parameters on the glucose detection efficiency were evaluated. Practical application of AuNPs-nSi MS glucose analysis in urine samples was also demonstrated in this study.