Science.gov

Sample records for reference tissue quantification

  1. Dual time point method for the quantification of irreversible tracer kinetics: A reference tissue approach applied to [(18)F]-FDOPA brain PET.

    PubMed

    Alves, Isadora L; Meles, Sanne K; Willemsen, Antoon Tm; Dierckx, Rudi A; Marques da Silva, Ana M; Leenders, Klaus L; Koole, Michel

    2016-01-01

    The Patlak graphical analysis (PGAREF) for quantification of irreversible tracer binding with a reference tissue model was approximated by a dual time point imaging approach (DTPREF). The DTPREF was applied to 18 [(18)F]-FDOPA brain scans using the occipital cortex as reference region (DTPOCC) and compared to both PGAOCC and striatal-to-occipital ratios (SOR). Pearson correlation analysis and Bland-Altman plots showed an excellent correlation and good agreement between DTPOCC and PGAOCC, while correlations between SOR and PGAOCC were consistently lower. Linear discriminant analysis (LDA) demonstrated a similar performance for all methods in differentiating patients with Parkinson's disease (PD) from healthy controls (HC). Specifically for [(18)F]-FDOPA brain imaging, these findings validate DTPOCC as an approximation for PGAOCC, providing the same quantitative information while reducing the acquisition time to two short static scans. For PD patients, this approach can greatly improve patient comfort while reducing motion artifacts and increasing image quality. In general, DTPREF can improve the clinical applicability of tracers with irreversible binding characteristics when a reference tissue is available.

  2. QUANTIFICATION OF TISSUE PROPERTIES IN SMALL VOLUMES

    SciTech Connect

    J. MOURANT; ET AL

    2000-12-01

    The quantification of tissue properties by optical measurements will facilitate the development of noninvasive methods of cancer diagnosis and detection. Optical measurements are sensitive to tissue structure which is known to change during tumorigenesis. The goals of the work presented in this paper were to verify that the primary scatterers of light in cells are structures much smaller than the nucleus and then to develop an optical technique that can quantify parameters of structures the same size as the scattering features in cells. Polarized, elastic back-scattering was found to be able to quantify changes in scattering properties for turbid media consisting of scatterers of the size found in tissue.

  3. Ultrasound strain imaging for quantification of tissue function: cardiovascular applications

    NASA Astrophysics Data System (ADS)

    de Korte, Chris L.; Lopata, Richard G. P.; Hansen, Hendrik H. G.

    2013-03-01

    With ultrasound imaging, the motion and deformation of tissue can be measured. Tissue can be deformed by applying a force on it and the resulting deformation is a function of its mechanical properties. Quantification of this resulting tissue deformation to assess the mechanical properties of tissue is called elastography. If the tissue under interrogation is actively deforming, the deformation is directly related to its function and quantification of this deformation is normally referred as `strain imaging'. Elastography can be used for atherosclerotic plaques characterization, while the contractility of the heart or skeletal muscles can be assessed with strain imaging. We developed radio frequency (RF) based ultrasound methods to assess the deformation at higher resolution and with higher accuracy than commercial methods using conventional image data (Tissue Doppler Imaging and 2D speckle tracking methods). However, the improvement in accuracy is mainly achieved when measuring strain along the ultrasound beam direction, so 1D. We further extended this method to multiple directions and further improved precision by using compounding of data acquired at multiple beam steered angles. In arteries, the presence of vulnerable plaques may lead to acute events like stroke and myocardial infarction. Consequently, timely detection of these plaques is of great diagnostic value. Non-invasive ultrasound strain compounding is currently being evaluated as a diagnostic tool to identify the vulnerability of plaques. In the heart, we determined the strain locally and at high resolution resulting in a local assessment in contrary to conventional global functional parameters like cardiac output or shortening fraction.

  4. Determination of reference microRNAs for relative quantification in grass carp (Ctenopharyngodon idella).

    PubMed

    Xu, Xiao-Yan; Shen, Yu-Bang; Fu, Jian-Jun; Lu, Li-Qun; Li, Jia-Le

    2014-02-01

    Relative quantification is the strategy of choice for processing real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) data in microRNA (miRNA) expression studies. Normalization of relative quantification data is performed by comparison to reference genes. In teleost species, such as grass carp (Ctenopharyngodon idella), the determination of reference miRNAs and the optimal numbers of these that should be used has not been widely studied. In the present study, the stability of seven miRNAs (miR-126-3p, miR-101a, miR-451, miR-22a, miR-146, miR-142a-5p and miR-192) was investigated by RT-qPCR in different tissues and in different development stages of grass carp. Stability values were calculated with geNorm, NormFinder, BestKeeper and Delta CT algorithms. The results showed that tissue type is an important variability factor for miRNA expression stability. All seven miRNAs had good stability values and, therefore, could be used as reference miRNAs. When all tissues and developmental stages were considered, miR-101a was the most stable miRNA. When each tissue type was considered separately, the most stable miRNAs were 126-3p in blood and liver, 101a in the gills, 192 in the kidney, 451 in the intestine and 22a in the brain, head kidney, spleen, heart, muscle, skin and fin. 126-3p was the most stable reference miRNA gene during developmental stages 1-5, while 22a was the most stable during developmental stages 6-18. Overall, this study provides valuable information about the reference miRNAs that can be used to perform appropriate normalizations when undertaking relative quantification in RT-qPCR studies of grass carp.

  5. Image quantification of high-throughput tissue microarray

    NASA Astrophysics Data System (ADS)

    Wu, Jiahua; Dong, Junyu; Zhou, Huiyu

    2006-03-01

    Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time and provides valuable information on expression of proteins within tissues at a cellular and sub-cellular level. TMA technology overcomes the bottleneck of traditional tissue analysis and allows it to catch up with the rapid advances in lead discovery. Studies using TMA on immunohistochemistry (IHC) can produce a large amount of images for interpretation within a very short time. Manual interpretation does not allow accurate quantitative analysis of staining to be undertaken. Automatic image capture and analysis has been shown to be superior to manual interpretation. The aims of this work is to develop a truly high-throughput and fully automated image capture and analysis system. We develop a robust colour segmentation algorithm using hue-saturation-intensity (HSI) colour space to provide quantification of signal intensity and partitioning of staining on high-throughput TMA. Initial segmentation results and quantification data have been achieved on 16,000 TMA colour images over 23 different tissue types.

  6. Quantification of Carbohydrates in Grape Tissues Using Capillary Zone Electrophoresis

    PubMed Central

    Zhao, Lu; Chanon, Ann M.; Chattopadhyay, Nabanita; Dami, Imed E.; Blakeslee, Joshua J.

    2016-01-01

    Soluble sugars play an important role in freezing tolerance in both herbaceous and woody plants, functioning in both the reduction of freezing-induced dehydration and the cryoprotection of cellular constituents. The quantification of soluble sugars in plant tissues is, therefore, essential in understanding freezing tolerance. While a number of analytical techniques and methods have been used to quantify sugars, most of these are expensive and time-consuming due to complex sample preparation procedures which require the derivatization of the carbohydrates being analyzed. Analysis of soluble sugars using capillary zone electrophoresis (CZE) under alkaline conditions with direct UV detection has previously been used to quantify simple sugars in fruit juices. However, it was unclear whether CZE-based methods could be successfully used to quantify the broader range of sugars present in complex plant extracts. Here, we present the development of an optimized CZE method capable of separating and quantifying mono-, di-, and tri-saccharides isolated from plant tissues. This optimized CZE method employs a column electrolyte buffer containing 130 mM NaOH, pH 13.0, creating a current of 185 μA when a separation voltage of 10 kV is employed. The optimized CZE method provides limits-of-detection (an average of 1.5 ng/μL) for individual carbohydrates comparable or superior to those obtained using gas chromatography–mass spectrometry, and allows resolution of non-structural sugars and cell wall components (structural sugars). The optimized CZE method was successfully used to quantify sugars from grape leaves and buds, and is a robust tool for the quantification of plant sugars found in vegetative and woody tissues. The increased analytical efficiency of this CZE method makes it ideal for use in high-throughput metabolomics studies designed to quantify plant sugars. PMID:27379118

  7. Mouse spleen tissue as a staining intensity reference for immunohistochemistry.

    PubMed

    Moon, Yeonsook; Park, Gyeongsin; Han, Kyungja; Kang, Chang-Suk; Lee, Wonbae

    2008-01-01

    Immunohistochemistry (IHC) is widely used in diagnostic practice and research, but it is limited due to its subjective nature and weakness in reproducibility. For successful interpretation, IHC requires an internal reference system that controls for procedural variables and provides a staining intensity reference. We investigated the feasibility of using mouse spleen tissue as an intensity reference in conventional IHC. Formalin-fixed, paraffin-embedded mouse (BALB/c) spleen tissue was stained with variable procedural conditions including primary antibody (Ab) types, antigen retrieval methods, chromogen exposure times, and secondary Ab concentrations. Mouse spleen tissue showed identical staining intensity regardless of primary Ab types, even without primary Ab, and showed minimal differences according to retrieval methods. However, it showed various staining intensities according to chromogen exposure time and secondary Ab concentration. When mouse spleen was included in tissue microarrays and compared with the c-erbB2 IHC scoring system, splenic B cells showed weak membrane staining compatible with score 1+, whereas splenic plasma cells showed strong staining intensity compatible with score 3+. These results show that mouse spleen tissue can serve as a staining intensity reference for the interpretation of IHC.

  8. Reference genes for quantitative PCR in the adipose tissue of mice with metabolic disease.

    PubMed

    Almeida-Oliveira, Fernanda; Leandro, João G B; Ausina, Priscila; Sola-Penna, Mauro; Majerowicz, David

    2017-04-01

    Obesity and diabetes are metabolic diseases and they are increasing in prevalence. The dynamics of gene expression associated with these diseases is fundamental to identifying genes involved in related biological processes. qPCR is a sensitive technique for mRNA quantification and the most commonly used method in gene-expression studies. However, the reliability of these results is directly influenced by data normalization. As reference genes are the major normalization method used, this work aims to identify reference genes for qPCR in adipose tissues of mice with type-I diabetes or obesity. We selected 12 genes that are commonly used as reference genes. The expression of these genes in the adipose tissues of mice was analyzed in the context of three different experimental protocols: 1) untreated animals; 2) high-fat-diet animals; and 3) streptozotocin-treated animals. Gene-expression stability was analyzed using four different algorithms. Our data indicate that TATA-binding protein is stably expressed across adipose tissues in control animals. This gene was also a useful reference when the brown adipose tissues of control and obese mice were analyzed. The mitochondrial ATP synthase F1 complex gene exhibits stable expression in subcutaneous and perigonadal adipose tissue from control and obese mice. Moreover, this gene is the best reference for qPCR normalization in adipose tissue from streptozotocin-treated animals. These results show that there is no perfect stable gene suited for use under all experimental conditions. In conclusion, the selection of appropriate genes is a prerequisite to ensure qPCR reliability and must be performed separately for different experimental protocols.

  9. Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues

    PubMed Central

    Mangeot-Peter, Lauralie; Legay, Sylvain; Hausman, Jean-Francois; Esposito, Sergio; Guerriero, Gea

    2016-01-01

    Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp. PMID:27649158

  10. Evaluation of the reliability of maize reference assays for GMO quantification.

    PubMed

    Papazova, Nina; Zhang, David; Gruden, Kristina; Vojvoda, Jana; Yang, Litao; Buh Gasparic, Meti; Blejec, Andrej; Fouilloux, Stephane; De Loose, Marc; Taverniers, Isabel

    2010-03-01

    A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb

  11. A regularized full reference tissue model for PET neuroreceptor mapping.

    PubMed

    Mandeville, Joseph B; Sander, Christin Y M; Wey, Hsiao-Ying; Hooker, Jacob M; Hansen, Hanne D; Svarer, Claus; Knudsen, Gitte M; Rosen, Bruce R

    2016-06-27

    The full reference tissue model (FRTM) is a PET analysis framework that includes both free and specifically bound compartments within tissues, together with rate constants defining association and dissociation from the specifically bound compartment. The simplified reference tissue model (SRTM) assumes instantaneous exchange between tissue compartments, and this "1-tissue" approximation reduces the number of parameters and enables more robust mapping of non-displaceable binding potentials. Simulations based upon FRTM have shown that SRTM exhibits biases that are spatially dependent, because biases depend upon binding potentials. In this work, we describe a regularized model (rFRTM) that employs a global estimate of the dissociation rate constant from the specifically bound compartment (k4). The model provides an internal calibration for optimizing k4 through the reference-region outflow rate k2', a model parameter that should be a global constant but varies regionally in SRTM. Estimates of k4 by rFRTM are presented for four PET radioligands. We show that SRTM introduces bias in parameter estimates by assuming an infinite value for k4, and that rFRTM ameliorates bias with an appropriate choice of k4. Theoretical considerations and simulations demonstrate that rFRTM reduces bias in non-displaceable binding potentials. A two-parameter reduction of the model (rFRTM2) provides robust mapping at a voxel-wise level. With a structure similar to SRTM, the model is easily implemented and can be applied as a PET reference region analysis that reduces parameter bias without substantially altering parameter variance.

  12. Quantification of tissue oxygenation levels using diffuse reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

    B. S., Suresh Anand; N., Sujatha

    2010-12-01

    Tumor growth is characterized by increased metabolic activity. The light absorption profile of hemoglobin in dysplastic tissue is different from a normal tissue. Neovascularization is a hallmark of many diseases and can serve as a predictive biomarker for the detection of cancers. Spectroscopic techniques can provide information about the metabolic and morphological changes related to the progression of neoplasia. Diffuse reflectance spectroscopy (DRS) measures the absorption and scattering properties of a biological tissue and this method can provide clinically useful information for the early diagnosis of epithelial precancers. We used tissue simulating phantoms with absorbing and scattering molecules for the determination of total hemoglobin concentration, hemoglobin oxygen saturation and intensity difference between the deoxy and oxy hemoglobin bands. The results show promising approach for the differentiating normal and malignant states of a tissue.

  13. Quantification of tissue oxygenation levels using diffuse reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

    B. S., Suresh Anand; N., Sujatha

    2011-08-01

    Tumor growth is characterized by increased metabolic activity. The light absorption profile of hemoglobin in dysplastic tissue is different from a normal tissue. Neovascularization is a hallmark of many diseases and can serve as a predictive biomarker for the detection of cancers. Spectroscopic techniques can provide information about the metabolic and morphological changes related to the progression of neoplasia. Diffuse reflectance spectroscopy (DRS) measures the absorption and scattering properties of a biological tissue and this method can provide clinically useful information for the early diagnosis of epithelial precancers. We used tissue simulating phantoms with absorbing and scattering molecules for the determination of total hemoglobin concentration, hemoglobin oxygen saturation and intensity difference between the deoxy and oxy hemoglobin bands. The results show promising approach for the differentiating normal and malignant states of a tissue.

  14. Quantification of petroleum-type hydrocarbons in avian tissue

    USGS Publications Warehouse

    Gay, M.L.; Belisle, A.A.; Patton, J.F.

    1980-01-01

    Summary: Methods were developed for the analysis of 16 hydrocarbons in avian tissue. Mechanical extraction with pentane was followed by clean-up on Florisil and Silicar. Residues were determined by gas--liquid chromatography and gas-liquid, chromatography-mass spectrometry. The method was applied to the analysis of liver, kidney, fat, and brain tissue of mallard ducks (Anas platyrhynchos) fed a mixture of hydrocarbons. Measurable concentrations of all compounds analyzed were present in all tissues except brain. Highest concentrations were in fat.

  15. Evaluation of the ERETIC method as an improved quantitative reference for 1H HR-MAS spectroscopy of prostate tissue.

    PubMed

    Albers, Mark J; Butler, Thomas N; Rahwa, Iman; Bao, Nguyen; Keshari, Kayvan R; Swanson, Mark G; Kurhanewicz, John

    2009-03-01

    The Electronic REference To access In vivo Concentrations (ERETIC) method was applied to (1)H HR-MAS spectroscopy. The accuracy, precision, and stability of ERETIC as a quantitative reference were evaluated in solution and human prostate tissue samples. For comparison, the reliability of 3-(trimethylsilyl)propionic-2,2,3,3-d(4) acid (TSP) as a quantitation reference was also evaluated. The ERETIC and TSP peak areas were found to be stable in solution over the short-term and long-term, with long-term relative standard deviations (RSDs) of 4.10% and 2.60%, respectively. Quantification of TSP in solution using the ERETIC peak as a reference and a calibrated, rotor-dependent conversion factor yielded results with a precision < or =2.9% and an accuracy error < or =4.2% when compared with the expected values. The ERETIC peak area reproducibility was superior to TSP's reproducibility, corrected for mass, in both prostate surgical and biopsy samples (4.53% vs. 21.2% and 3.34% vs. 31.8%, respectively). Furthermore, the tissue TSP peaks exhibited only 27.5% of the expected area, which would cause an overestimation of metabolite concentrations if used as a reference. The improved quantification accuracy and precision provided by ERETIC may enable the detection of smaller metabolic differences that may exist between individual tissue samples and disease states.

  16. Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

    PubMed Central

    2010-01-01

    Background Quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, Bactrocera dorsalis (Hendel). Results Two different programs, geNorm and Normfinder, were used to analyze the data. According to geNorm, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by Normfinder are quite the same as the results with geNorm; α-TUB is always one of the most stable genes in each sample validated by the two programs. Conclusions In this study, we validated the suitable reference genes for gene expression profiling in different tissues of B. dorsalis. Moreover, appropriate reference genes were selected out for gene expression profiling of the

  17. Quantification of tissue texture with photoacoustic spectrum analysis

    NASA Astrophysics Data System (ADS)

    Wang, Xueding; Xu, Guan; Meng, Zhuo-Xian; Lin, Jiandie; Carson, Paul

    2014-05-01

    Photoacoustic (PA) imaging is an emerging technology that could map the functional contrasts in deep biological tissues in high resolution by "listening" to the laser induced thermoelastic waves. Almost all of the current studies in PA imaging are focused on the intensity of the PA signals as an indication of the optical absorbance of the biological tissues. Our group has for the first time demonstrated that the frequency domain power distribution of the broadband PA signals encode the texture information within the regions-of-interest (ROI). Following the similar method of ultrasound spectral analysis (USSA), photoacoustic spectrum analysis (PASA) could evaluate the relative concentrations and, more importantly, the dimensions of microstructures of the optically absorbing materials in biological tissues, including lipid, collagen, water and hemoglobin. By providing valuable insights into tissue pathology, PASA should benefit basic research and clinical management of many diseases, and may help achieve eventual "noninvasive biopsy". In this work, taking advantage of the optical absorption contrasts contributed by lipid and hemoglobin at 1200-nm and 532-nm wavelengths respectively, we investigated the capability of PASA in identifying histological changes corresponding to fat accumulation livers through the study on ex vivo and in situ mouse models. The PA signals from the mouse livers were acquired using our PA and US dual-modality imaging system, and analyzed in the frequency domain. After quantifying the power spectrum by fitting it to a first order model, three spectral parameters, including the intercept, the midband fit and the slope, were extracted and used to differentiate fatty livers from normal livers. The comparison between the PASA parameters from the normal and the fatty livers supports our hypotheses that PASA can quantitatively identify the microstructure changes in liver tissues for differentiating normal and fatty livers.

  18. Automatic Segmentation and Quantification of White and Brown Adipose Tissues from PET/CT Scans.

    PubMed

    Hussein, Sarfaraz; Green, Aileen; Watane, Arjun; Reiter, David; Chen, Xinjian; Papadakis, Georgios Z; Wood, Bradford; Cypess, Aaron; Osman, Medhat; Bagci, Ulas

    2016-12-06

    In this paper, we investigate the automatic detection of white and brown adipose tissues using Positron Emission Tomography/ Computed Tomography (PET/CT) scans, and develop methods for the quantification of these tissues at the whole-body and body-region levels. We propose a patient-specific automatic adiposity analysis system with two modules. In the first module, we detect white adipose tissue (WAT) and its two sub-types from CT scans: Visceral Adipose Tissue (VAT) and Subcutaneous Adipose Tissue (SAT). This process relies conventionally on manual or semi-automated segmentation, leading to inefficient solutions. Our novel framework addresses this challenge by proposing an unsupervised learning method to separate VAT from SAT in the abdominal region for the clinical quantification of central obesity. This step is followed by a context driven label fusion algorithm through sparse 3D Conditional Random Fields (CRF) for volumetric adiposity analysis. In the second module, we automatically detect, segment, and quantify brown adipose tissue (BAT) using PET scans because unlike WAT, BAT is metabolically active. After identifying BAT regions using PET, we perform a co-segmentation procedure utilizing asymmetric complementary information from PET and CT. Finally, we present a new probabilistic distance metric for differentiating BAT from non-BAT regions. Both modules are integrated via an automatic body-region detection unit based on one-shot learning. Experimental evaluations conducted on 151 PET/CT scans achieve state-of-the-art performances in both central obesity as well as brown adiposity quantification.

  19. Quantification of Histamine and Carcinine in Drosophila melanogaster Tissues.

    PubMed

    Denno, Madelaine E; Privman, Eve; Borman, Ryan P; Wolin, Danielle C; Venton, B Jill

    2016-03-16

    Histamine is a neurotransmitter crucial to the visual processing of Drosophila melanogaster. It is inactivated by metabolism to carcinine, a β-alanyl derivative, and the same enzyme that controls that process also converts dopamine to N-β-alanyl-dopamine. Direct detection of histamine and carcinine has not been reported in single Drosophila brains. Here, we quantify histamine, carcinine, dopamine, and N-β-alanyl-dopamine in Drosophila tissues by capillary electrophoresis coupled to fast-scan cyclic voltammetry (CE-FSCV). Limits of detection were low, 4 ± 1 pg for histamine, 10 ± 4 pg for carcinine, 2.8 ± 0.3 pg for dopamine, and 9 ± 3 pg for N-β-alanyl-dopamine. Tissue content was compared in the brain, eyes, and cuticle from wild-type (Canton S) and mutant (tan(3) and ebony(1)) strains. In tan(3) mutants, the enzyme that produces histamine from carcinine is nonfunctional, whereas in ebony(1) mutants, the enzyme that produces carcinine from histamine is nonfunctional. In all fly strains, the neurotransmitter content was highest in the eyes and there were no strain differences for tissue content in the cuticle. The main finding was that carcinine levels changed significantly in the mutant flies, whereas histamine levels did not. In particular, tan(3) flies had significantly higher carcinine levels in the eyes and brain than Canton S or ebony(1) flies. N-β-Alanyl-dopamine was detected in tan(3) mutants but not in other strains. These results show the utility of CE-FSCV for sensitive detection of histamine and carcinine, which allows a better understanding of their content and metabolism in different types of tissues to be obtained.

  20. Quantification of human body fat tissue percentage by MRI.

    PubMed

    Müller, Hans-Peter; Raudies, Florian; Unrath, Alexander; Neumann, Heiko; Ludolph, Albert C; Kassubek, Jan

    2011-01-01

    The MRI-based evaluation of the quantity and regional distribution of adipose tissue is one objective measure in the investigation of obesity. The aim of this article was to report a comprehensive and automatic analytical method for the determination of the volumes of subcutaneous fat tissue (SFT) and visceral fat tissue (VFT) in either the whole human body or selected slices or regions of interest. Using an MRI protocol in an examination position that was convenient for volunteers and patients with severe diseases, 22 healthy subjects were examined. The software platform was able to merge MRI scans of several body regions acquired in separate acquisitions. Through a cascade of image processing steps, SFT and VFT volumes were calculated. Whole-body SFT and VFT distributions, as well as fat distributions of defined body slices, were analysed in detail. Complete three-dimensional datasets were analysed in a reproducible manner with as few operator-dependent interventions as possible. In order to determine the SFT volume, the ARTIS (Adapted Rendering for Tissue Intensity Segmentation) algorithm was introduced. The advantage of the ARTIS algorithm was the delineation of SFT volumes in regions in which standard region grow techniques fail. Using the ARTIS algorithm, an automatic SFT volume detection was feasible. MRI data analysis was able to determine SFT and VFT volume percentages using new analytical strategies. With the techniques described, it was possible to detect changes in SFT and VFT percentages of the whole body and selected regions. The techniques presented in this study are likely to be of use in obesity-related investigations, as well as in the examination of longitudinal changes in weight during various medical conditions.

  1. Quantification of the edge effect in calcified bioprosthetic tissues.

    PubMed

    Wika, K E; Utoh, J; Brown, J; Harasaki, H

    1993-10-01

    In bioprosthetic tissue samples that had been implanted in the subcutaneous space of rats, and recurring pattern of calcification was observed. In this pattern, which we call the edge effect, the interior of the tissue is calcified and is surrounded and separated from the subcutaneous fluid by a zone that is free from calcification. The edge effect has been qualitatively described in the literature for subcutaneous implants and for valve leaflets, and it may be related to the mechanism of calcification for these materials. The thickness of the calcification free outer layer was quantified for glutaraldehyde treated bovine pericardium, glycerol treated bovine pericardium, glutaraldehyde treated human dura mater, and glycerol treated human dura mater. The edge effect values were found to be unique and consistent for each material type, and they were inversely related to the shrinkage temperatures and the calcium contents of the materials. It was determined that the chemical treatment was more important than the tissue type in determining the edge effect value.

  2. Proposal for C-Hordein as Reference Material in Gluten Quantification.

    PubMed

    Huang, Xin; Kanerva, Päivi; Salovaara, Hannu; Stoddard, Frederick L; Sontag-Strohm, Tuula

    2017-03-15

    The concentration of residual barley prolamin (hordein) in gluten-free products is overestimated by the R5 ELISA method when calibrated against the wheat gliadin standard. The reason for this may be that the composition of the gliadin standard is different from the composition of hordeins. This study showed that the recognition of whole hordein by R5 antibody mainly came from C-hordein, which is more reactive than the other hordeins. The proportion of C-hordein in total hordein ranged from 16 to 33% of common Finnish barley cultivars used in this study and was always higher than that of ω-gliadin, the homologous protein class in the gliadin standard, which may account for the overestimation. Thus, a hordein standard is needed for barley prolamin quantification instead of the gliadin standard. When gluten-free oat flour was spiked with barley flour, the prolamin concentration was overestimated 1.8-2.5 times with the gliadin standard, whereas estimates in the correct range were obtained when the standard was 40% C-hordein mixed with an inert protein. A preparative-scale method was developed to isolate and purify C-hordein, and C-hordein is proposed as a reference material to calibrate barley prolamin quantification in R5-based assays.

  3. Certification of methylmercury content in two fresh-frozen reference materials: SRM 1947 Lake Michigan fish tissue and SRM 1974b organics in mussel tissue (Mytilus edulis).

    PubMed

    Davis, W Clay; Christopher, S J; Pugh, Rebecca S; Donard, O F X; Krupp, Eva A; Point, David; Horvat, Milena; Gibicar, D; Kljakovic-Gaspic, Z; Porter, Barbara J; Schantz, Michele M

    2007-04-01

    This paper describes the development of two independent analytical methods for the extraction and quantification of methylmercury from marine biota. The procedures involve microwave extraction, followed by derivatization and either headspace solid-phase microextraction (SPME) with a polydimethylsiloxane (PDMS)-coated silica fiber or back-extraction into iso-octane. The identification and quantification of the extracted compounds is carried out by capillary gas chromatography/mass spectrometric (GC/MS) and inductively coupled plasma mass spectrometric (GC/ICP-MS) detection. Both methods were validated for the determination of methylmercury (MeHg) concentrations in a variety of biological standard reference materials (SRMs) including fresh-frozen tissue homogenates of SRM 1946 Lake Superior fish tissue and SRM 1974a organics in mussel tissue (Mytilus edulis) and then applied to the certification effort of SRM 1947 Lake Michigan fish tissue and SRM 1974b organics in mussel tissue (Mytilus edulis). While past certifications of methylmercury in tissue SRMs have been based on two independent methods from the National Institute of Standards and Technology (NIST) and participating laboratories, the methods described within provide improved protocols and will allow future certification efforts to be based on at least two independent analytical methods within NIST.

  4. Quantification of pork, chicken and beef by using a novel reference molecule.

    PubMed

    Sakai, Yumiko; Kotoura, Satoshi; Yano, Takeo; Kurihara, Takashi; Uchida, Kouji; Miake, Kiyotaka; Akiyama, Hiroshi; Tanabe, Soichi

    2011-01-01

    A standard plasmid was constructed as a novel reference molecule for use in real-time quantitative PCR assays to verify the identity of beef, pork, chicken, mutton, and horseflesh. The plasmid contained a target domain of the cytochrome b (cyt b) gene and an artificial DNA sequence. Primers CO-F and CO-R, and probe CO-P were specifically designed to detect the artificial sequence. The calculated R² values of the standard curves (10³-10⁷ copies per reaction) for the five species ranged between 0.998 and 0.999 in the quantification analysis. The constructed plasmid provides a universal method for measuring the copy number of cyt b DNA in minced meat. This method would be a useful procedure for verifying food labels.

  5. Quantification of progesterone binding in mammary tissue of pregnant ewes

    SciTech Connect

    Smith, J.J.; Capuco, A.V.; Akers, R.M.

    1987-06-01

    Progestin-binding sites in mammary tissue from 14 prepartum, multiparous ewes at 50, 80, 115, and 140 d of gestation were demonstrated by the binding of (/sup 3/H) R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) to ovine mammary cytosol in the presence of sodium molybdate and excess cortisol. Homogenization extracted 89% of total mammary receptors (nuclear) into cytosol. Binding was specific for progestins and was of high affinity. The average dissociation constant for (/sup 3/H) R5020 specifically bound to receptors extracted into mammary cytosol was 1.9 (+/- .4) x 10/sup -9/ M (n = 14) and did not change significantly over the test period. However, binding capacities (fmol/mg cytosolic protein) differed according to stage of gestation with averages of 125 +/- 53, 149 +/- 26, 656 +/- 216, 57 +/- 22 at 50, 80, 115, and 140 d of pregnancy, respectively. Increased number of progestin-binding sites at 115 d of gestation (whether data are expressed per unit of tissue weight, DNA, or cytosolic protein) suggests that an increase per mammary epithelial cell may be necessary to produce the full lobuloalveolar proliferation observed at this stage of gestation.

  6. Quantification of pelvic soft tissue artifact in multiple static positions.

    PubMed

    Hara, Reiko; Sangeux, Morgan; Baker, Richard; McGinley, Jennifer

    2014-02-01

    Soft tissue artifact (STA) has been identified as the most critical source of error in clinical gait analysis. Multiple calibration is a technique to reduce the impact of STA on kinematic data, which involves several static calibrations through the range of motion of the joint of interest. This study investigated how skin markers at the pelvis were displaced in relation to anatomical body landmarks in multiple static calibration positions. The magnitude and direction of the pelvic marker displacement was assessed in nine different body positions including 90° and 45° hip flexion, maximum hip extension, and pelvic tilt in 20 healthy young adults. ASIS markers were found to be more susceptible to relative displacement than PSIS markers, with displacement particularly evident in positions where the hip was flexed (up to 17 mm). A strong correlation was found between the hip flexion angle and marker displacement (r(2) = 0.70). While the estimated impact of pelvic STA on gait kinematics was relatively small, the findings suggest that activities with large hip flexion would cause larger STA with a greater impact on pelvic kinematics. The skin surface located over the ASIS differed by a mean of 17 mm between standing and supine positions, which could affect the inter-ASIS distance and the location of hip joint center (HJC) by up to 20mm and 10mm, respectively.

  7. Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees.

    PubMed

    Gadiou, S; Kundu, J K

    2012-06-01

    A SYBR Green(®)-based one step RT-qPCR assay was developed for the detection and quantification of Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). The RT-qPCR assay employed seven plant-expressed genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, ubiquitin, ribosomal protein S19, Rubisco, RNA polymerase subunit II and β-actin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. Idared, Champion, Fragrance). The average expression stability (M) found by GeNorm software suggest that GAPDH and S19 were the most stable reference genes. We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR.

  8. Fully automatic and nonparametric quantification of adipose tissue in fat-water separation MR imaging.

    PubMed

    Wang, Defeng; Shi, Lin; Chu, Winnie C W; Hu, Miao; Tomlinson, Brian; Huang, Wen-Hua; Wang, Tianfu; Heng, Pheng Ann; Yeung, David K W; Ahuja, Anil T

    2015-11-01

    Despite increasing demand and research efforts, currently there is no consensus on the protocol for automated and reliable quantification of adipose tissue (AT) and visceral adipose tissue (VAT) using MRI. The purpose of this study was to propose a novel computational method with enhanced objectiveness for the quantification of AT and VAT in fat-water separation MRI. 3T data from IDEAL were acquired for the fat-water separation. Fat tissues were separated from nonfat regions (background air, bone, water, and other nonfat tissues) using K-means clustering (K = 2). From the binary fat mask, arm regions were separated from body based on the relative size of connected component. AT was obtained from the binary body fat mask. With the initial contour as the outer boundary of body fat, the subcutaneous adipose tissue (SAT) and VAT were separated using deformable model driven by a specifically generated deformation field pointing to the inner boundary of SAT. The proposed method was tested on 16 patients with dyslipidemia and evaluated by comparing the correlation with semi-automatic segmentation results. Good robustness was also observed in the proposed method from the Bland-Altman plots. Compared to other established fat segmentation methods, the proposed method is highly objective for fat-water separation MRI with minimal variability induced by subjective parameter settings.

  9. Fat emulsions as diffusive reference standards for tissue simulating phantoms?

    PubMed

    Di Ninni, Paola; Bérubé-Lauzière, Yves; Mercatelli, Luca; Sani, Elisa; Martelli, Fabrizio

    2012-10-20

    Intralipid 20% was recently suggested as a diffusive reference standard for tissue simulating phantoms. In this work, we extend previously obtained results to other fat emulsions, specifically Intralipid 10%, Intralipid 30%, Lipovenoes 10%, Lipovenoes 10% PhosphoLipid Reduced, Lipovenoes 20%, Lipofundin S 10%, and Lipofundin S 20%. Of particular importance for practical applications, our measurements carried out at a wavelength of 751 nm show the following features. First, these products show high stability and small batch-to-batch variations in their diffusive optical properties, similar to Intralipid 20%. Second, the absorption coefficient of Intralipid, Lipovenoes, and Lipofundin S are very similar and their measured values are within the experimental errors; moreover the reduced scattering coefficient of Intralipid 20%, Lipovenoes 20%, and Lipofundin S 20% are similar and their measured values are within 5%. Third, the reduced scattering coefficient of Intralipid 10% and Intralipid 30% can be scaled from that of Intralipid 20% with an error of 9% and 2%, respectively. A similar scaling property is valid for Lipovenoes and Lipofundin S. We have verified that this scaling property depends on the composition of the fat emulsions: If the ingredients exactly scale with the concentration then the reduced scattering coefficient almost exactly scale as well.

  10. Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes.

    PubMed

    Wang, Y-K; Li, X; Song, Z-Q; Yang, C-X

    2017-04-13

    To ensure accurate normalization and quantification of target RNA transcripts using reverse transcription quantitative polymerase chain reaction (RT-qPCR), most studies focus on the identification of stably expressed gene(s) as internal reference. However, RNA preparation methods could also be an important factor, especially for test samples of limited quantity (e.g. oocytes). In this study, we aimed to select appropriate reference gene(s), and evaluate the effect of RNA preparation methods on gene expression quantification in porcine oocytes and cumulus cells during in vitro maturation. Expression profiles of seven genes (GAPDH, 18S, YWHAG, BACT, RPL4, HPRT1 and PPIA) were examined, on RNA samples extracted from cumulus cells (RNeasy Kit) and oocytes (RNeasy Kit and Lysis Kit) during in vitro maturation, respectively. Interestingly, different RNA preparation methods were found to potentially affect the quantification of reference gene expression in pig oocytes cultured in vitro. After geNorm analyses, the most suitable genes for normalization were identified, GAPDH/18S for cumulus cells and YWHAG/BACT for oocytes, respectively. Thus, our results provide useful data and information on the selection of better reference genes and RNA preparation method for related functional studies.

  11. ID-SERS Based Reference Method for Quantification of Large Biomolecules on a Single Chip

    NASA Astrophysics Data System (ADS)

    Yaghobian, Fatemeh; Stosch, Rainer; Henrion, André; Güttler, Bernd

    2010-08-01

    Accuracy and precision of quantitative SERS results have been significantly increased by applying a method based on the so-called isotope-dilution (ID) principle. In this ID-SERS approach, an isotopically labeled analogue of the target molecule (isotopologue) is spiked to the sample at a known concentration. Due to the slight difference in their molar masses, some Raman bands of the heavier isotopologue are red-shifted with respect to the same signals resulting from the unlabelled compound. As a result, spectra evaluation is reduced to the determination of intensity ratios rather than absolute intensities, and the unknown quantity of the analyte can be calculated from the known quantity of the standard. This procedure is of particular interest in the development of highly accurate reference procedures for metrology in chemistry. Because the sample is spiked prior to any further treatment, potential loss of material or matrix effects would equally affect both isotopologues, without influencing the final result. The method has been successfully applied for quantifying small diagnostic marker molecules like creatinine at their relevant serum concentration levels using silver colloids as SERS substrates. Now, the ID-SERS approach has been realized as a "one-chip" approach using "Bio-chips" made of intrinsically grown spherical silver nanoparticles with gaps less than 10 nm in between (Fig. 1). In addition, the scope of the method has been extended to larger biomolecules like peptides which will be shown using the example of the human growth-hormone (hGH) peptide T12 at physiologically relevant serum concentration levels (Fig. 2). Further developments towards the quantification of full proteins will also be reported.

  12. ID-SERS Based Reference Method for Quantification of Large Biomolecules on a Single Chip

    SciTech Connect

    Yaghobian, Fatemeh; Stosch, Rainer; Henrion, Andre; Guettler, Bernd

    2010-08-06

    Accuracy and precision of quantitative SERS results have been significantly increased by applying a method based on the so-called isotope-dilution (ID) principle. In this ID-SERS approach, an isotopically labeled analogue of the target molecule (isotopologue) is spiked to the sample at a known concentration. Due to the slight difference in their molar masses, some Raman bands of the heavier isotopologue are red-shifted with respect to the same signals resulting from the unlabelled compound. As a result, spectra evaluation is reduced to the determination of intensity ratios rather than absolute intensities, and the unknown quantity of the analyte can be calculated from the known quantity of the standard. This procedure is of particular interest in the development of highly accurate reference procedures for metrology in chemistry. Because the sample is spiked prior to any further treatment, potential loss of material or matrix effects would equally affect both isotopologues, without influencing the final result. The method has been successfully applied for quantifying small diagnostic marker molecules like creatinine at their relevant serum concentration levels using silver colloids as SERS substrates. Now, the ID-SERS approach has been realized as a 'one-chip' approach using 'Bio-chips' made of intrinsically grown spherical silver nanoparticles with gaps less than 10 nm in between (Fig. 1). In addition, the scope of the method has been extended to larger biomolecules like peptides which will be shown using the example of the human growth-hormone (hGH) peptide T12 at physiologically relevant serum concentration levels (Fig. 2). Further developments towards the quantification of full proteins will also be reported.

  13. White adipose tissue reference network: a knowledge resource for exploring health-relevant relations.

    PubMed

    Kelder, Thomas; Summer, Georg; Caspers, Martien; van Schothorst, Evert M; Keijer, Jaap; Duivenvoorde, Loes; Klaus, Susanne; Voigt, Anja; Bohnert, Laura; Pico, Catalina; Palou, Andreu; Bonet, M Luisa; Dembinska-Kiec, Aldona; Malczewska-Malec, Malgorzata; Kieć-Wilk, Beata; Del Bas, Josep M; Caimari, Antoni; Arola, Lluis; van Erk, Marjan; van Ommen, Ben; Radonjic, Marijana

    2015-01-01

    Optimal health is maintained by interaction of multiple intrinsic and environmental factors at different levels of complexity-from molecular, to physiological, to social. Understanding and quantification of these interactions will aid design of successful health interventions. We introduce the reference network concept as a platform for multi-level exploration of biological relations relevant for metabolic health, by integration and mining of biological interactions derived from public resources and context-specific experimental data. A White Adipose Tissue Health Reference Network (WATRefNet) was constructed as a resource for discovery and prioritization of mechanism-based biomarkers for white adipose tissue (WAT) health status and the effect of food and drug compounds on WAT health status. The WATRefNet (6,797 nodes and 32,171 edges) is based on (1) experimental data obtained from 10 studies addressing different adiposity states, (2) seven public knowledge bases of molecular interactions, (3) expert's definitions of five physiologically relevant processes key to WAT health, namely WAT expandability, Oxidative capacity, Metabolic state, Oxidative stress and Tissue inflammation, and (4) a collection of relevant biomarkers of these processes identified by BIOCLAIMS ( http://bioclaims.uib.es ). The WATRefNet comprehends multiple layers of biological complexity as it contains various types of nodes and edges that represent different biological levels and interactions. We have validated the reference network by showing overrepresentation with anti-obesity drug targets, pathology-associated genes and differentially expressed genes from an external disease model dataset. The resulting network has been used to extract subnetworks specific to the above-mentioned expert-defined physiological processes. Each of these process-specific signatures represents a mechanistically supported composite biomarker for assessing and quantifying the effect of interventions on a

  14. Preclinical In vivo Imaging for Fat Tissue Identification, Quantification, and Functional Characterization

    PubMed Central

    Marzola, Pasquina; Boschi, Federico; Moneta, Francesco; Sbarbati, Andrea; Zancanaro, Carlo

    2016-01-01

    Localization, differentiation, and quantitative assessment of fat tissues have always collected the interest of researchers. Nowadays, these topics are even more relevant as obesity (the excess of fat tissue) is considered a real pathology requiring in some cases pharmacological and surgical approaches. Several weight loss medications, acting either on the metabolism or on the central nervous system, are currently under preclinical or clinical investigation. Animal models of obesity have been developed and are widely used in pharmaceutical research. The assessment of candidate drugs in animal models requires non-invasive methods for longitudinal assessment of efficacy, the main outcome being the amount of body fat. Fat tissues can be either quantified in the entire animal or localized and measured in selected organs/regions of the body. Fat tissues are characterized by peculiar contrast in several imaging modalities as for example Magnetic Resonance Imaging (MRI) that can distinguish between fat and water protons thank to their different magnetic resonance properties. Since fat tissues have higher carbon/hydrogen content than other soft tissues and bones, they can be easily assessed by Computed Tomography (CT) as well. Interestingly, MRI also discriminates between white and brown adipose tissue (BAT); the latter has long been regarded as a potential target for anti-obesity drugs because of its ability to enhance energy consumption through increased thermogenesis. Positron Emission Tomography (PET) performed with 18F-FDG as glucose analog radiotracer reflects well the metabolic rate in body tissues and consequently is the technique of choice for studies of BAT metabolism. This review will focus on the main, non-invasive imaging techniques (MRI, CT, and PET) that are fundamental for the assessment, quantification and functional characterization of fat deposits in small laboratory animals. The contribution of optical techniques, which are currently regarded with

  15. X-ray fluorescence and energy dispersive x-ray diffraction for the quantification of elemental concentrations in breast tissue.

    PubMed

    Geraki, K; Farquharson, M J; Bradley, D A

    2004-01-07

    This paper presents improvements on a previously reported method for the measurement of elements in breast tissue specimens (Geraki et al 2002 Phys. Med. Biol. 47 2327-39). A synchrotron-based system was used for the detection of the x-ray fluorescence (XRF) emitted from iron, copper, zinc and potassium in breast tissue specimens, healthy and cancerous. Calibration models resulting from the irradiation of standard aqueous solutions were used for the quantification of the elements. The present developments concentrate on increasing the convergence between the tissue samples and the calibration models, therefore improving accuracy. For this purpose the composition of the samples in terms of adipose and fibrous tissue was evaluated, using an energy dispersive x-ray diffraction (EDXRD) system. The relationships between the attenuation and scatter properties of the two tissue components and water were determined through Monte Carlo simulations. The results from the simulations and the EDXRD measurements allowed the XRF data from each specimen to be corrected according to its composition. The statistical analysis of the elemental concentrations of the different groups of specimens reveals that all four elements are found in elevated levels in the tumour specimens. The increase is less pronounced for iron and copper and most for potassium and zinc. Other observed features include the substantial degree of inhomogeneity of elemental distributions within the volume of the specimens, varying between 4% and 36% of the mean, depending on the element and the type of the sample. The accuracy of the technique, based on the measurement of a standard reference material, proved to be between 3% and 22% depending on the element, which presents only a marginal improvement (1%-3%) compared to the accuracy of the previously reported results. The measurement precision was between 1% and 9% while the calculated uncertainties on the final elemental concentrations ranged between 10% and 16%.

  16. 18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

    SciTech Connect

    Kim, Jong Seo; Fillmore, Thomas L.; Liu, Tao; Robinson, Errol W.; Hossain, Mahmud; Champion, Boyd L.; Moore, Ronald J.; Camp, David G.; Smith, Richard D.; Qian, Weijun

    2011-10-11

    Selected reaction monitoring-mass spectrometry (SRM-MS) is an emerging technology for high throughput targeted protein quantification and verification in biological and biomarker discovery studies; however, the cost associated with the use of stable isotope labeled synthetic peptides as internal standards is prohibitive for quantitatively screening large numbers of candidate proteins as often required in the pre-verification phase of biomarker discovery. Herein we present the proof-of-concept experiments of using an 18O-labeled 'universal' reference as comprehensive internal standards for quantitative SRM-MS analysis. With an 18O-labeled whole proteome sample as reference, every peptide of interest will have its own corresponding heavy isotope labeled internal standard, thus providing an ideal approach for quantitative screening of a large number of candidates using SRM-MS. Our results showed that the 18O incorporation efficiency using a recently improved protocol was >99.5% for most peptides investigated, a level comparable to 13C/15N labeled synthetic peptides in terms of heavy isotope incorporation. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into mouse plasma with an 18O-labeled mouse plasma reference. A dynamic range of four orders of magnitude in relative concentration was obtained with high reproducibility (i.e., coefficient of variance <10%) based on the 16O/18O peak area ratios. Absolute and relative quantification of C-reactive protein and prostate-specific antigen were demonstrated by coupling an 18O-labeled reference with standard additions of protein standards. Collectively, our results demonstrated that the use of 18O-labeled reference provides a convenient and effective strategy for quantitative SRM screening of large number of candidate proteins.

  17. Wheat Germ Agglutinin Staining as a Suitable Method for Detection and Quantification of Fibrosis in Cardiac Tissue after Myocardial Infarction

    PubMed Central

    Emde, B.; Heinen, A.; Gödecke, A.; Bottermann, K.

    2014-01-01

    The quantification of fibrotic tissue is an important task in the analysis of cardiac remodeling. The use of established fibrosis staining techniques is limited on frozen cardiac tissue sections due to a reduced color contrast compared to paraffin embedded sections. We therefore used FITC-labeled wheat germ agglutinin (WGA), which marks fibrotic tissue in comparable quality as the established picrosirius red (SR) staining, for the staining of post myocardial infarction scar tissue. The fibrosis amount was quantified in a histogram-based approach using the non-commercial image processing program ImageJ. Our results clearly demonstrate that WGA-FITC is a suitable marker for cardiac fibrosis in frozen tissue sections. In combination with the histogram-based analysis, this new quantification approach is i) easy and fast to perform; ii) suitable for raw frozen tissue sections; and iii) allows the use of additional antibodies in co-immunostaining. PMID:25578975

  18. Tissue-dependent VEGF and GLUT1 induction in a rat hemorrhage model: With regard to diagnostic application of mRNA quantification in forensic pathology.

    PubMed

    Zhao, Dong; Michiue, Tomomi; Maeda, Hitoshi

    2015-10-01

    Systemic hypoxia is inevitably involved in the death process to a varying extent. Hypoxia-response factors proved useful in forensic pathology in previous studies; however, fundamental investigations using animal models are expected to reinforce the findings from autopsy practice. An animal experiment using a rat model of fixed-volume hemorrhage was performed to apply basic insight into quantitative mRNA analyses in forensic pathology. Male Sprague-Dawley rats (n=5) were anesthetized, bled from the femoral artery (24ml/kg; about 30% of total circulating blood volume), and decapitated after 1 or 2h. Tissue samples of the heart, brain (hippocampus), kidney, liver, lung and skeletal muscle were collected for RNA and protein analyses. Quantitative analyses of VEGF, GLUT1 and GAPDH mRNAs were performed with TaqMan real-time RT-PCR assay. In the sham control without bleeding, mRNA quantification revealed the tissue-dependent mRNA levels in physiological condition. Relative quantification of VEGF and GLUT1 showed significant inductions under hemorrhage at the mRNA level, using GAPDH as endogenous reference. In conclusion, tissue-dependent induction patterns of VEGF and GLUT1 were revealed in the volume-fixed hemorrhage rat model. This study could practically guide the selection of mRNA markers and tissue samples in forensic pathology related to tissue ischemia and cellular hypoxia for autopsy cases.

  19. Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

    PubMed

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

  20. Point-of-care device for quantification of bilirubin in skin tissue.

    PubMed

    Alla, Suresh K; Huddle, Adam; Butler, Joshua D; Bowman, Peggy S; Clark, Joseph F; Beyette, Fred R

    2011-03-01

    Steady state diffuse reflectance spectroscopy is a nondestructive method for obtaining biochemical and physiological information from skin tissue. In medical conditions such as neonatal jaundice excess bilirubin in the blood stream diffuses into the surrounding tissue leading to a yellowing of the skin. Diffuse reflectance measurement of the skin tissue can provide real time assessment of the progression of a disease or a medical condition. Here we present a noninvasive point-of-care system that utilizes diffuse reflectance spectroscopy to quantifying bilirubin from skin reflectance spectra. The device consists of an optical system integrated with a signal processing algorithm. The device is then used as a platform to study two different spectral databases. The first spectral database is a jaundice animal model in which the jaundice reflectance spectra are synthesized from normal skin. The second spectral database is the spectral measurements collected on human volunteers to quantify the different chromophores and other physical properties of the tissue such as Hematocrit, Hemoglobin, etc. The initial trials from each of these spectral databases have laid the foundation to verify the performance of this bilirubin quantification device.

  1. Identification of Reliable Reference Genes for Quantification of MicroRNAs in Serum Samples of Sulfur Mustard-Exposed Veterans

    PubMed Central

    Gharbi, Sedigheh; Shamsara, Mehdi; Khateri, Shahriar; Soroush, Mohammad Reza; Ghorbanmehr, Nassim; Tavallaei, Mahmood; Nourani, Mohammad Reza; Mowla, Seyed Javad

    2015-01-01

    Objective In spite of accumulating information about pathological aspects of sulfur mustard (SM), the precise mechanism responsible for its effects is not well understood. Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims. Materials and Methods In this case and control experimental study, using quantitative real-time polymerase chain reaction (qRT-PCR), we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war (1980-1988) and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle (Cq) method were employed to find the least variable reference gene. Results miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that. Conclusion We demonstrate that non-miRNA reference genes have the least stabil- ity in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers. PMID:26464821

  2. Absolute Quantification of Lipophilic Shellfish Toxins by Quantitative Nuclear Magnetic Resonance Using Removable Internal Reference Substance with SI Traceability.

    PubMed

    Kato, Tsuyoshi; Saito, Maki; Nagae, Mika; Fujita, Kazuhiro; Watai, Masatoshi; Igarashi, Tomoji; Yasumoto, Takeshi; Inagaki, Minoru

    2016-01-01

    Okadaic acid (OA), a lipophilic shellfish toxin, was accurately quantified using quantitative nuclear magnetic resonance with internal standards for the development of an authentic reference standard. Pyridine and the residual proton in methanol-d4 were used as removable internal standards to limit any contamination. They were calibrated based on a maleic acid certified reference material. Thus, the concentration of OA was traceable to the SI units through accurate quantitative NMR with an internal reference substance. Signals from the protons on the oxygenated and unsaturated carbons of OA were used for quantification. A reasonable accuracy was obtained by integrating between the lower and upper (13)C satellite signal range when more than 4 mg of OA was used. The best-determined purity was 97.4% (0.16% RSD) when 20 mg of OA was used. Dinophysistoxin-1, a methylated analog of OA having an almost identical spectrum, was also quantified by using the same methodology.

  3. Quantification of DNA Extracted from Formalin Fixed Paraffin-Embeded Tissue Comparison of Three Techniques: Effect on PCR Efficiency

    PubMed Central

    Panigrahi, Manoj Kumar; Suryavanshi, Moushumi; Mehta, Anurag; Saikia, Kandarpa Kumar

    2016-01-01

    Introduction Mutation detection from Formalin Fixed Paraffin-Embedding (FFPE) tissue in molecular lab became a necessary tool for defining potential targeted drug. Accurate quantification of DNA extracted from FFPE tissue is necessary for downstream applications like Polymerase Chain Reaction (PCR), sequencing etc. Aim To check and define which method for FFPE DNA quantification is suitable for downstream processes. Materials and Methods In this experimental experience study Biorad Smartspec Plus spectrophotomery, Qubit Fluorometer, and Qiagen Rotorgene qPCR was used to compare 20 FFPE DNA quantification in Rajiv Gandhi Cancer Institute and Research Centre, in 2015 and quantified amount of DNA used for PCR reaction. Results The average concentration of DNA extracted from FFPE tissue measured using the spectrophotometer was much higher than the concentration measured using the Qubit Fluorometer and qPCR. Conclusion Results varied depending upon the technique used. A fluorometric analysis may be more suitable for quantification of DNA samples extracted from FFPE tissue compared with spectrophotometric analysis. But qPCR is the best technique because it details DNA quantity along with quality of amplifiable DNA from FFPE tissue. PMID:27790419

  4. Optimization of supervised cluster analysis for extracting reference tissue input curves in (R)-[11C]PK11195 brain PET studies

    PubMed Central

    Yaqub, Maqsood; van Berckel, Bart NM; Schuitemaker, Alie; Hinz, Rainer; Turkheimer, Federico E; Tomasi, Giampaolo; Lammertsma, Adriaan A; Boellaard, Ronald

    2012-01-01

    Performance of two supervised cluster analysis (SVCA) algorithms for extracting reference tissue curves was evaluated to improve quantification of dynamic (R)-[11C]PK11195 brain positron emission tomography (PET) studies. Reference tissues were extracted from images using both a manually defined cerebellum and SVCA algorithms based on either four (SVCA4) or six (SVCA6) kinetic classes. Data from controls, mild cognitive impairment patients, and patients with Alzheimer's disease were analyzed using various kinetic models including plasma input, the simplified reference tissue model (RPM) and RPM with vascular correction (RPMVb). In all subject groups, SVCA-based reference tissue curves showed lower blood volume fractions (Vb) and volume of distributions than those based on cerebellum time-activity curve. Probably resulting from the presence of specific signal from the vessel walls that contains in normal condition a significant concentration of the 18 kDa translocation protein. Best contrast between subject groups was seen using SVCA4-based reference tissues as the result of a lower number of kinetic classes and the prior removal of extracerebral tissues. In addition, incorporation of Vb in RPM improved both parametric images and binding potential contrast between groups. Incorporation of Vb within RPM, together with SVCA4, appears to be the method of choice for analyzing cerebral (R)-[11C]PK11195 neurodegeneration studies. PMID:22588187

  5. Optimization of supervised cluster analysis for extracting reference tissue input curves in (R)-[(11)C]PK11195 brain PET studies.

    PubMed

    Yaqub, Maqsood; van Berckel, Bart N M; Schuitemaker, Alie; Hinz, Rainer; Turkheimer, Federico E; Tomasi, Giampaolo; Lammertsma, Adriaan A; Boellaard, Ronald

    2012-08-01

    Performance of two supervised cluster analysis (SVCA) algorithms for extracting reference tissue curves was evaluated to improve quantification of dynamic (R)-[(11)C]PK11195 brain positron emission tomography (PET) studies. Reference tissues were extracted from images using both a manually defined cerebellum and SVCA algorithms based on either four (SVCA4) or six (SVCA6) kinetic classes. Data from controls, mild cognitive impairment patients, and patients with Alzheimer's disease were analyzed using various kinetic models including plasma input, the simplified reference tissue model (RPM) and RPM with vascular correction (RPMV(b)). In all subject groups, SVCA-based reference tissue curves showed lower blood volume fractions (V(b)) and volume of distributions than those based on cerebellum time-activity curve. Probably resulting from the presence of specific signal from the vessel walls that contains in normal condition a significant concentration of the 18 kDa translocation protein. Best contrast between subject groups was seen using SVCA4-based reference tissues as the result of a lower number of kinetic classes and the prior removal of extracerebral tissues. In addition, incorporation of V(b) in RPM improved both parametric images and binding potential contrast between groups. Incorporation of V(b) within RPM, together with SVCA4, appears to be the method of choice for analyzing cerebral (R)-[(11)C]PK11195 neurodegeneration studies.

  6. Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

    PubMed Central

    Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

  7. Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications.

    PubMed

    Sfakis, Lauren; Kamaldinov, Tim; Larsen, Melinda; Castracane, James; Khmaladze, Alexander

    2016-11-01

    Quantifying confocal images to enable location of specific proteins of interest in three-dimensional (3D) is important for many tissue engineering (TE) applications. Quantification of protein localization is essential for evaluation of specific scaffold constructs for cell growth and differentiation for application in TE and tissue regeneration strategies. Although obtaining information regarding protein expression levels is important, the location of proteins within cells grown on scaffolds is often the key to evaluating scaffold efficacy. Functional epithelial cell monolayers must be organized with apicobasal polarity with proteins specifically localized to the apical or basolateral regions of cells in many organs. In this work, a customized program was developed using the LabVIEW platform to quantify protein positions in Z-stacks of confocal images of epithelial cell monolayers. The program's functionality is demonstrated through salivary gland TE, since functional salivary epithelial cells must correctly orient many proteins on the apical and basolateral membranes. Bio-LabVIEW Image Matrix Evaluation (Bio-LIME) takes 3D information collected from confocal Z-stack images and processes the fluorescence at each pixel to determine cell heights, nuclei heights, nuclei widths, protein localization, and cell count. As a demonstration of its utility, Bio-LIME was used to quantify the 3D location of the Zonula occludens-1 protein contained within tight junctions and its change in 3D position in response to chemical modification of the scaffold with laminin. Additionally, Bio-LIME was used to demonstrate that there is no advantage of sub-100 nm poly lactic-co-glycolic acid nanofibers over 250 nm fibers for epithelial apicobasal polarization. Bio-LIME will be broadly applicable for quantification of proteins in 3D that are grown in many different contexts.

  8. Analysis of dissected tissues with digital holographic microscopy: quantification of inflammation mediated tissue alteration, influence of sample preparation, and reliability of numerical autofocusing

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

    2015-03-01

    Quantitative phase imaging with digital holographic microscopy (DHM) allows label-free imaging of tissue sections and quantification of the spatial refractive index distribution, which is of interest for applications in digital pathology. We show that DHM allows quantitative imaging of different layers in unstained tissue samples by detection of refractive index changes. In addition, we evaluate the automated refocussing feature of DHM for application on dissected tissues and could achieve highly reproducible holographic autofocusing for unstained and moderately stained samples. Finally, it is demonstrated that in human ulcerative colitis patients the average tissue refractive index is reduced significantly in all parts of the inflamed colonic wall in comparison to patients in remission.

  9. Quantification of in vivo fluorescence decoupled from the effects of tissue optical properties using fiber-optic spectroscopy measurements

    NASA Astrophysics Data System (ADS)

    Kim, Anthony; Khurana, Mamta; Moriyama, Yumi; Wilson, Brian C.

    2010-11-01

    We present a method for tissue fluorescence quantification in situ using a handheld fiber optic probe that measures both the fluorescence and diffuse reflectance spectra. A simplified method to decouple the fluorescence spectrum from distorting effects of the tissue optical absorption and scattering is developed, with the objective of accurately quantifying the fluorescence in absolute units. The primary motivation is measurement of 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) concentration in tissue during fluorescence-guided resection of malignant brain tumors. This technique is validated in phantoms and ex vivo mouse tissues, and tested in vivo in a rabbit brain tumor model using ALA-PpIX fluorescence contrast.

  10. Non-Invasive, Simultaneous Quantification of Vascular Oxygenation and Glucose Uptake in Tissue

    PubMed Central

    Rajaram, Narasimhan; Reesor, Andrew F.; Mulvey, Christine S.; Frees, Amy E.; Ramanujam, Nirmala

    2015-01-01

    We report the development of non-invasive, fiber-based diffuse optical spectroscopy for simultaneously quantifying vascular oxygenation (SO2) and glucose uptake in solid tumors in vivo. Glucose uptake was measured using a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). Quantification of label-free SO2 and 2-NBDG-fluorescence-based glucose uptake 60 minutes after administration of the tracer (2-NBDG60) was performed using computational models of light-tissue interaction. This study was carried out on normal tissue and 4T1 and 4T07 murine mammary tumor xenografts in vivo. Injection of 2-NBDG did not cause a significant change in optical measurements of SO2, demonstrating its suitability as a functional reporter of tumor glucose uptake. Correction of measured 2-NBDG-fluorescence for the effects of absorption and scattering significantly improved contrast between tumor and normal tissue. The 4T1 and 4T07 tumors showed significantly decreased SO2, and 4T1 tumors demonstrated increased 2-NBDG60 compared with normal tissue (60 minutes after the administration of 2-NBDG when perfusion-mediated effects have cleared). 2-NBDG-fluorescence was found to be highly sensitive to food deprivation-induced reduction in blood glucose levels, demonstrating that this endpoint is indeed sensitive to glycolytic demand. 2-NBDG60 was also found to be linearly related to dose, underscoring the importance of calibrating for dose when comparing across animals or experiments. 4T1 tumors demonstrated an inverse relationship between 2-NBDG60 and SO2 that was consistent with the Pasteur effect, particularly when exposed to hypoxic gas breathing. Our results illustrate the potential of optical spectroscopy to provide valuable information about the metabolic status of tumors, with important implications for cancer prognosis. PMID:25635865

  11. A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues.

    PubMed

    Penning, Louis C; Vrieling, Henriette E; Brinkhof, Bas; Riemers, Frank M; Rothuizen, Jan; Rutteman, Gerard R; Hazewinkel, Herman A W

    2007-12-15

    For a proper determination of relative mRNA expression levels with real-time quantitative PCR (Q-PCR) internal standards, such as the expression of reference genes, are of utmost importance. For cats, in contrast to dogs, no validation of reference genes has been published. Our goal was to evaluate frequently used reference genes for the analysis of relative mRNA levels from feline tissues in a SYBR Green-based Q-PCR protocol. First, primers were optimized on mRNA-derived cDNA from liver and kidney tissues of randomly chosen (healthy and diseased) cats. Then, the expression variation and stability of each reference gene within a specific tissue was determined. Dental roots and crowns, heart (left ventricle), renal, liver, lung, and mammary gland tissues from 3 to 11 cats of different breeds, sexes, ages, and disease status were included in this study. Averaging relative stabilities over these six tissues revealed the usefulness of each tested gene as reference gene. In order to compensate for the expression variation of a reference gene within a specific tissue, as much as six reference genes (e.g. RPL17, RPL30, RPS7, YWHAZ, and HPRT) were required to obtain highly reliable data in cat tissues. The optimal set of reference genes depended on the tissue analyzed and should, ideally, be selected and evaluated at the start of each experimental condition. A comparison with a similar evaluation in dogs revealed three issues: (i) most ribosomal genes are suitable in both species; (ii) good non-ribosomal reference genes differ; (iii) more feline than canine reference genes are required for proper analysis.

  12. Bovine muscle 20S proteasome. III: Quantification in tissue crude extracts using ELISA and radial immunodiffusion techniques and practical applications.

    PubMed

    Aubry, L; Sentandreu, M A; Levieux, D; Ouali, A; Dutaud, D

    2006-10-01

    The 20S proteasome is a large complex (700kDa) that exhibits endo- and exo-peptidase activities with wide specificity. In postmortem muscles, several sets of evidence suggest a possible significant contribution of proteasome to meat tenderisation. Hence, an accurate and rapid quantification procedure is needed to attest that new function during the ageing of meat. In the present work, we developed an ELISA test enabling the quantification of nM concentrations of the 20S proteasome. We further tested the radial immunodiffusion (RID) technique described as a more simple method that can quantitatively determine the concentration of an antigen in a complex mixture. The ELISA test allowed us to quantify the 20S protesome in tissue homogenates and fluids with a recovery of 100%, a coefficient of variation lower than 5% and a detection limit of 9ng/ml. Quantification of the 20S proteasome in various bovine tissue by ELISA showed the highest concentration in liver followed by spleen and kidney, with muscles exhibiting the lowest concentrations. In addition, measurement of the proteasome concentration in eight different bovine muscles with various metabolic profiles led to the conclusion that the relationship between muscle metabolic properties and proteasome concentration is rather complex. Nevertheless, heart muscle exhibited the highest proteasome content (331μg/g wet tissue) whereas the lowest values were found for M. Tensor Fascia Latae (213μg/g wet tissue), a fast twitch white muscle, M. Supraspinatus (209μg/g wet tissue), a slow twitch red muscle and M. Pectoralis profondus (203μg/g wet tissue), an intermediate muscle. As compared to other endogenous peptidases, muscle tissue contains relatively high amounts of proteasome. Hence this complex can be quantified using the RID, which allows quantification of protein in the μg range. Plotting the concentration values determined with both methods for all bovine tissues tested gave a straight line with a correlation

  13. Quantification of effects of cancer on elastic properties of breast tissue by Atomic Force Microscopy.

    PubMed

    Ansardamavandi, Arian; Tafazzoli-Shadpour, Mohammad; Omidvar, Ramin; Jahanzad, Iisa

    2016-07-01

    Different behaviors of cells such as growth, differentiation and apoptosis widely differ in case of diseases. The mechanical properties of cells and tissues can be used as a clue for diagnosis of pathological conditions. Here, we implemented Atomic Force Microscopy to evaluate the extent of alteration in mechanical stiffness of tissue layers from patients affected by breast cancer and investigated how data can be categorized based on pathological observations. To avoid predefined categories, Fuzzy-logic algorithm as a novel method was used to divide and categorize the derived Young׳s modulus coefficients (E). Such algorithm divides data among groups in such way that data of each group are mostly similar while dissimilar with other groups. The algorithm was run for different number of categories. Results showed that three (followed by two with small difference) groups categorized data best. Three categories were defined as (E<3000Pa, 30007000Pa) among which data were allocated. The first cluster was assumed as the cellular region while the last cluster was referred to the fibrous parts of the tissue. The intermediate region was due to other non-cellular parts. Results indicated 50% decline of average Young׳s modulus of cellular region of cancerous tissues compared to healthy tissues. The average Young׳s modulus of non-cellular area of normal tissues was slightly lower than that of cancerous tissues, although the difference was not statistically different. Through clustering, the measured Young׳s moduli of different locations of cancerous tissues, a quantified approach was developed to analyze changes in elastic modulus of a spectrum of components of breast tissue which can be applied in diagnostic mechanisms of cancer development, since in cancer progression the softening cell body facilitates the migration of cancerous cells through the original tumor and endothelial junctions.

  14. Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata

    NASA Astrophysics Data System (ADS)

    Pu, Fei; Yang, Bingye; Ke, Caihuan

    2015-07-01

    Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 ( ACT-2), elongation factor 1 alpha ( EF-1α), elongation factor 1 beta ( EF-1β), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), ubiquitin ( UBQ), β-tubulin ( β-TUB), and 18S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene ( Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ and β-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further functional genomics studies in this economically valuable marine bivalve.

  15. Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues

    PubMed Central

    Tomaszewski, Bartłomiej; De Prins, Sofie; Jacobs, Griet; Koppen, Gudrun; Mathers, John C.; Langie, Sabine A. S.

    2013-01-01

    DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the “gold standard” of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases. PMID:24260150

  16. In vivo quantification of motion in liver parenchyma and its application in shistosomiasis tissue characterization

    NASA Astrophysics Data System (ADS)

    Badawi, Ahmed M.; Hashem, Ahmed M.; Youssef, Abou-Bakr M.; Abdel-Wahab, Mohamed F.

    1995-03-01

    Schistosomiasis is a major problem in Egypt, despite an active control program it is estimated to exist in about 1/3 of the population. Deposition of less functioning fibrous tissues in the liver is the major contributory factor to the hepatic pathology. Fibrous tissues consist of a complex array of connective matrix material and a variety of collagen isotopes. As a result of an increased stromal density (collagen content), the parenchyma became more ectogenic and less elastic (hard). In this study we investigated the effect of cardiac mechanical impulses from the heart and aorta on the kinetics of the liver parenchyma. Under conditions of controlled patient movements and suspended respiration, a 30 frame per second of 588 X 512 ultrasound images (cineloop, 32 pels per cm) are captured from an aTL ultrasound machine then digitized. The image acquisition is triggered by the R wave of the ECG of the patient. The motion that has a forced oscillation form in the liver parenchyma is quantified by tracking of small box (20 - 30 pels) in 16 directions for all the successive 30 frames. The tracking was done using block matching techniques (the max correlation between boxes in time, frequency domains, and the minimum SAD (sum absolute difference) between boxes). The motion is quantified for many regions at different positions within the liver parenchyma for 80 cases of variable degrees of schisto., cirrhotic livers, and for normal livers. The velocity of the tissue is calculated from the displacement (quantified motion), time between frames, and the scan time for the ultrasound scanner. We found that the motion in liver parenchyma is small in the order of very few millimeters, and the attenuation of the mechanical wave for one ECG cycle is higher in the schisto. and cirrhotic livers than in the normal ones. Finally quantification of motion in liver parenchyma due to cardiac impulses under controlled limb movement and respiration may be of value in the characterization of

  17. Quantification of Calcified Particles in Human Valve Tissue Reveals Asymmetry of Calcific Aortic Valve Disease Development

    PubMed Central

    Yabusaki, Katsumi; Hutcheson, Joshua D.; Vyas, Payal; Bertazzo, Sergio; Body, Simon C.; Aikawa, Masanori; Aikawa, Elena

    2016-01-01

    Recent studies indicated that small calcified particles observable by scanning electron microscopy (SEM) may initiate calcification in cardiovascular tissues. We hypothesized that if the calcified particles precede gross calcification observed in calcific aortic valve disease (CAVD), they would exhibit a regional asymmetric distribution associated with CAVD development, which always initiates at the base of aortic valve leaflets adjacent to the aortic outflow in a region known as the fibrosa. Testing this hypothesis required counting the calcified particles in histological sections of aortic valve leaflets. SEM images, however, do not provide high contrast between components within images, making the identification and quantification of particles buried within tissue extracellular matrix difficult. We designed a new unique pattern-matching based technique to allow for flexibility in recognizing particles by creating a gap zone in the detection criteria that decreased the influence of non-particle image clutter in determining whether a particle was identified. We developed this flexible pattern particle-labeling (FpPL) technique using synthetic test images and human carotid artery tissue sections. A conventional image particle counting method (preinstalled in ImageJ) did not properly recognize small calcified particles located in noisy images that include complex extracellular matrix structures and other commonly used pattern-matching methods failed to detect the wide variation in size, shape, and brightness exhibited by the particles. Comparative experiments with the ImageJ particle counting method demonstrated that our method detected significantly more (p < 2 × 10−7) particles than the conventional method with significantly fewer (p < 0.0003) false positives and false negatives (p < 0.0003). We then applied the FpPL technique to CAVD leaflets and showed a significant increase in detected particles in the fibrosa at the base of the leaflets (p

  18. CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue

    NASA Astrophysics Data System (ADS)

    Corbisier, P.; Vincent, S.; Schimmel, H.; Kortekaas, A.-M.; Trapmann, S.; Burns, M.; Bushell, C.; Akgoz, M.; Akyürek, S.; Dong, L.; Fu, B.; Zhang, L.; Wang, J.; Pérez Urquiza, M.; Bautista, J. L.; Garibay, A.; Fuller, B.; Baoutina, A.; Partis, L.; Emslie, K.; Holden, M.; Chum, W. Y.; Kim, H.-H.; Phunbua, N.; Milavec, M.; Zel, J.; Vonsky, M.; Konopelko, L. A.; Lau, T. L. T.; Yang, B.; Hui, M. H. K.; Yu, A. C. H.; Viroonudomphol, D.; Prawettongsopon, C.; Wiangnon, K.; Takabatake, R.; Kitta, K.; Kawaharasaki, M.; Parkes, H.

    2012-01-01

    Key comparison CCQM-K86 was performed to demonstrate and document the capacity of interested national metrology institutes (NMIs) and designated institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a biological tissue. The study provides the support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length in the range of 70 to 100 nucleotides in a single genomic DNA extract from ground maize seed materials". The study was carried out under the auspices of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) and was piloted by the Institute for Reference Materials and Methods (IRMM) in Geel (Belgium). The following laboratories (in alphabetical order) participated in this key comparison: AIST (Japan), CENAM (Mexico), DMSc (Thailand), GLHK (Hong Kong), IRMM (European Union), KRISS (Republic of Korea), LGC (United Kingdom), MIRS/NIB (Slovenia), NIM (PR China), NIST (USA), NMIA (Australia), TÜBITAK UME (Turkey) and VNIIM (Russian Federation). The following laboratories (in alphabetical order) participated in a pilot study that was organized in parallel: LGC (United Kingdom), PKU (PR China), NFRI (Japan) and NIMT (Thailand). Good agreement was observed between the reported results of eleven participants. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

  19. Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues.

    PubMed

    Guelke, Eileen; Bucan, Vesna; Liebsch, Christina; Lazaridis, Andrea; Radtke, Christine; Vogt, Peter M; Reimers, Kerstin

    2015-04-10

    For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained.

  20. A method to obtain reference images for evaluation of ultrasonic tissue characterization techniques.

    PubMed

    Jensen, M S; Wilhjelm, J E; Sahl, B; Brandt, T; Martinsen, K; Jespersen, S K; Falk, E

    2002-05-01

    A general problem when evaluating ultrasonic methods for tissue characterization is that "a golden standard" is seldom known. This paper describes a manual method to obtain a reference image, with the same geometry as the ultrasound image, indicating spatial location of the different tissue types present in the biological tissue scanned in vitro. A 30 x 10 x 2 mm3 piece of formalin fixed porcine tissue was molded into an agar block, which on the top surface, contained a set of fiducial markers, spaced 2.5 mm. The block was submerged into 20 degrees C water and a set of parallel 7.5 MHz spatial compound ultrasound images of tissue and fiducial markers were recorded each 0.5 mm. Guided by the fiducial markers, the agar block was subsequently cut into slices 2.5 mm thick, photographed and finally analyzed histologically identifying these tissues: collagen rich, collagen poor, micro vessels and muscle fibres. Due to: (1) the cutting procedure, (2) the finite size of the ultrasound beam and (3) the spatial variation in propagation velocity, the macroscopic photographs did not align completely with the ultrasound images. Likewise, the histological image is a geometrically distorted version of the macroscopic photograph, due to the histological preparation process. The histological information was "mapped back" into the format of the ultrasound images the following way: On the macroscopic images, outlines were drawn manually which defined the border of the tissue. These outlines were superimposed on the corresponding ultrasound images (identified via the fiducial markers) and modified to encompass what appeared to be tissue regions on the ultrasound images and subsequently re-applied to the macroscopic image. This modified macroscopic outline was used as guideline when drawing outlines identifying regions of the various tissue types. Specifically, the macroscopic image revealed the borders between the different tissues, while the histological image identified the four

  1. Determination of reference genes for circadian studies in different tissues and mouse strains

    PubMed Central

    2010-01-01

    Background Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration. Results In the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons. Conclusions Genetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology. PMID:20712867

  2. Quantification of PrPC in bovine peripheral tissues: Analysis in wild-type and PrPC-deficient cattle.

    PubMed

    Kobayashi, Shin-Ichi; Ano, Yasuhisa; Sakudo, Akikazu; Yukawa, Masayoshi; Sigiura, Katsuaki; Manabe, Noboru; Nakayama, Hiroyuki; Onodera, Takashi

    2009-01-01

    Cellular PrP (PrPC) is necessary for bovine spongiform encephalopathy (BSE) infection. The purpose of the present experiment was the quantification of PrPC in peripheral tissues to assess the risk of BSE infection from these tissues. The tissue distribution of PrPC was examined by a sandwich enzyme-linked immunosorbent assay (sELISA) and histochemical analysis. PrPC-deficient cows were used as a negative control. The sELISA revealed that the brain contained the highest PrPC content (10.7 µg/g tissue), while other organs/tissues harbored lower amounts, in decreasing order as follows: longissimus capitis muscle, iliocostalis thoracis muscle, splenius muscle, biceps femoris muscle, triceps brachii muscle, longissimus thoracis muscle, ileum, jejunum, duodenum, colon, cecum, apex linguae, omotransversarius muscle, posterior part of the corpus linguae, anterior part of the corpus linguae and radix linguae (5.2- to 31-fold less PrPC than the brain). In the tissue/organs of PrP-deficient cows, PrPC levels were under the limit of detection. Histochemical analysis showed that PrPC was expressed in nerve cells in intestinal tissues. The presence of PrPC in the bovine tongue, skeletal muscles and intestines raises the possibility of PrPSc accumulation in these tissues, indicating that these organs/tissues may serve as potential sources of BSE infection.

  3. Validation of a Radiography-Based Quantification Designed to Longitudinally Monitor Soft Tissue Calcification in Skeletal Muscle

    PubMed Central

    Moore, Stephanie N.; Hawley, Gregory D.; Smith, Emily N.; Mignemi, Nicholas A.; Ihejirika, Rivka C.; Yuasa, Masato; Cates, Justin M. M.; Liu, Xulei; Schoenecker, Jonathan G.

    2016-01-01

    Introduction Soft tissue calcification, including both dystrophic calcification and heterotopic ossification, may occur following injury. These lesions have variable fates as they are either resorbed or persist. Persistent soft tissue calcification may result in chronic inflammation and/or loss of function of that soft tissue. The molecular mechanisms that result in the development and maturation of calcifications are uncertain. As a result, directed therapies that prevent or resorb soft tissue calcifications remain largely unsuccessful. Animal models of post-traumatic soft tissue calcification that allow for cost-effective, serial analysis of an individual animal over time are necessary to derive and test novel therapies. We have determined that a cardiotoxin-induced injury of the muscles in the posterior compartment of the lower extremity represents a useful model in which soft tissue calcification develops remote from adjacent bones, thereby allowing for serial analysis by plain radiography. The purpose of the study was to design and validate a method for quantifying soft tissue calcifications in mice longitudinally using plain radiographic techniques and an ordinal scoring system. Methods Muscle injury was induced by injecting cardiotoxin into the posterior compartment of the lower extremity in mice susceptible to developing soft tissue calcification. Seven days following injury, radiographs were obtained under anesthesia. Multiple researchers applied methods designed to standardize post-image processing of digital radiographs (N = 4) and quantify soft tissue calcification (N = 6) in these images using an ordinal scoring system. Inter- and intra-observer agreement for both post-image processing and the scoring system used was assessed using weighted kappa statistics. Soft tissue calcification quantifications by the ordinal scale were compared to mineral volume measurements (threshold 450.7mgHA/cm3) determined by μCT. Finally, sample-size calculations necessary

  4. Development of palm-based reference materials for the quantification of fatty acids composition.

    PubMed

    Tarmizi, Azmil Haizam Ahmad; Lin, Siew Wai; Kuntom, Ainie

    2008-01-01

    Characterisation of fatty acids composition of three palm-based reference materials was carried out through inter-laboratory proficiency tests. Twelve laboratories collaborated in these tests and the fatty acids compositions of palm oil, palm olein and palm stearin were determined by applying the MPOB Test Methods p3.4:2004 and p3.5:2004. Determination of consensus values and their uncertainties were based on the acceptable statistical agreement of results obtained from the collaborating laboratories. The consensus values and uncertainties (%) for each palm oil reference material produced are listed as follows : 0.20% (C12:0), 1.66+/-0.05% (C14:0), 43.39+/-0.39% (C16:0), 0.14+/-0.06% (C16:1), 3.90+/-0.11% (C18:0), 40.95+/-0.23% (C18:1), 9.68+/-0.21% (C18:2), 0.16+/-0.07% (C18:3) and 0.31+/-0.08% (C20:0) for fatty acids composition of palm oil; 0.23+/-0.04% (C12:0), 1.02+/-0.04% (C14:0), 39.66+/-0.19% (C16:0), 0.18+/-0.07% (C16:1), 3.81+/-0.04% (C18:0), 44.01+/-0.08% (C18:1), 10.73+/-0.08% (C18:2), 0.20+/-0.06% (C18:3) and 0.34+/-0.04% (C20:0) for fatty acids composition of palm olein; and 0.20% (C12:0), 1.14+/-0.05% (C14:0), 49.42+/-0.25% (C16:0), 0.16+/-0.08% (C16:1), 4.15+/-0.10% (C18:0), 36.14+/-0.77% (C18:1), 7.95+/-0.29% (C18:2), 0.11+/-0.07% (C18:3) and 0.30+/-0.08% (C20:0) for fatty acids composition of palm stearin.

  5. Reference-tissue correction of T2-weighted signal intensity for prostate cancer detection

    NASA Astrophysics Data System (ADS)

    Peng, Yahui; Jiang, Yulei; Oto, Aytekin

    2014-03-01

    The purpose of this study was to investigate whether correction with respect to reference tissue of T2-weighted MRimage signal intensity (SI) improves its effectiveness for classification of regions of interest (ROIs) as prostate cancer (PCa) or normal prostatic tissue. Two image datasets collected retrospectively were used in this study: 71 cases acquired with GE scanners (dataset A), and 59 cases acquired with Philips scanners (dataset B). Through a consensus histology- MR correlation review, 175 PCa and 108 normal-tissue ROIs were identified and drawn manually. Reference-tissue ROIs were selected in each case from the levator ani muscle, urinary bladder, and pubic bone. T2-weighted image SI was corrected as the ratio of the average T2-weighted image SI within an ROI to that of a reference-tissue ROI. Area under the receiver operating characteristic curve (AUC) was used to evaluate the effectiveness of T2-weighted image SIs for differentiation of PCa from normal-tissue ROIs. AUC (+/- standard error) for uncorrected T2-weighted image SIs was 0.78+/-0.04 (datasets A) and 0.65+/-0.05 (datasets B). AUC for corrected T2-weighted image SIs with respect to muscle, bladder, and bone reference was 0.77+/-0.04 (p=1.0), 0.77+/-0.04 (p=1.0), and 0.75+/-0.04 (p=0.8), respectively, for dataset A; and 0.81+/-0.04 (p=0.002), 0.78+/-0.04 (p<0.001), and 0.79+/-0.04 (p<0.001), respectively, for dataset B. Correction in reference to the levator ani muscle yielded the most consistent results between GE and Phillips images. Correction of T2-weighted image SI in reference to three types of extra-prostatic tissue can improve its effectiveness for differentiation of PCa from normal-tissue ROIs, and correction in reference to the levator ani muscle produces consistent T2-weighted image SIs between GE and Phillips MR images.

  6. Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.

    PubMed

    Steiner, Carine; Tille, Jean-Christophe; Lamerz, Jens; Kux van Geijtenbeek, Sabine; McKee, Thomas A; Venturi, Miro; Rubbia-Brandt, Laura; Hochstrasser, Denis; Cutler, Paul; Lescuyer, Pierre; Ducret, Axel

    2015-10-01

    The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry

  7. Epigenomic footprints across 111 reference epigenomes reveal tissue-specific epigenetic regulation of lincRNAs.

    PubMed

    Amin, Viren; Harris, R Alan; Onuchic, Vitor; Jackson, Andrew R; Charnecki, Tim; Paithankar, Sameer; Lakshmi Subramanian, Sai; Riehle, Kevin; Coarfa, Cristian; Milosavljevic, Aleksandar

    2015-02-18

    Tissue-specific expression of lincRNAs suggests developmental and cell-type-specific functions, yet tissue specificity was established for only a small fraction of lincRNAs. Here, by analysing 111 reference epigenomes from the NIH Roadmap Epigenomics project, we determine tissue-specific epigenetic regulation for 3,753 (69% examined) lincRNAs, with 54% active in one of the 14 cell/tissue clusters and an additional 15% in two or three clusters. A larger fraction of lincRNA TSSs is marked in a tissue-specific manner by H3K4me1 than by H3K4me3. The tissue-specific lincRNAs are strongly linked to tissue-specific pathways and undergo distinct chromatin state transitions during cellular differentiation. Polycomb-regulated lincRNAs reside in the bivalent state in embryonic stem cells and many of them undergo H3K27me3-mediated silencing at early stages of differentiation. The exquisitely tissue-specific epigenetic regulation of lincRNAs and the assignment of a majority of them to specific tissue types will inform future studies of this newly discovered class of genes.

  8. Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

    PubMed

    Chaouachi, Maher; Alaya, Akram; Ali, Imen Ben Haj; Hafsa, Ahmed Ben; Nabi, Nesrine; Bérard, Aurélie; Romaniuk, Marcel; Skhiri, Fethia; Saïd, Khaled

    2013-01-01

    KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

  9. Quantification of Arsenolipids in the Certified Reference Material NMIJ 7405-a (Hijiki) using HPLC/Mass Spectrometry after Chemical Derivatization

    PubMed Central

    2014-01-01

    Arsenic-containing lipids (arsenolipids) are novel natural products recently shown to be widespread in marine animals and algae. Research interest in these arsenic compounds lies in their possible role in the membrane chemistry of organisms and, because they occur in many popular seafoods, their human metabolism and toxicology. Progress has been restricted, however, by the lack of standard arsenolipids and of a quantitative method for their analysis. We report that the certified reference material CRM 7405-a (Hijiki) is a rich source of arsenolipids, and we describe a method based on HPLC-ICPMS/ESMS to quantitatively measure seven of the major arsenolipids present. Sample preparation involved extraction with DCM/methanol, a cleanup step with silica, and conversion of the (oxo)arsenolipids originally present to thio analogues by brief treatment with H2S. Compared to their oxo analogues, the thioarsenolipids showed much sharper peaks on reversed-phase HPLC, which facilitated their resolution and quantification. The compounds were determined by HPLC-ICPMS and HPLC-ESMS, which provided both arsenic-selective detection and high resolution molecular mass detection of the arsenolipids. In this way, the concentrations of two arsenic-containing hydrocarbons and five arsenosugar phospholipids are reported in the CRM Hijiki. This material may serve as a convenient source of characterized arsenolipids to delineate the presence of these compounds in seafoods and to facilitate research in a new era of arsenic biochemistry. PMID:25241916

  10. Quantification of arsenolipids in the certified reference material NMIJ 7405-a (Hijiki) using HPLC/mass spectrometry after chemical derivatization.

    PubMed

    Glabonjat, Ronald A; Raber, Georg; Jensen, Kenneth B; Ehgartner, Josef; Francesconi, Kevin A

    2014-10-21

    Arsenic-containing lipids (arsenolipids) are novel natural products recently shown to be widespread in marine animals and algae. Research interest in these arsenic compounds lies in their possible role in the membrane chemistry of organisms and, because they occur in many popular seafoods, their human metabolism and toxicology. Progress has been restricted, however, by the lack of standard arsenolipids and of a quantitative method for their analysis. We report that the certified reference material CRM 7405-a (Hijiki) is a rich source of arsenolipids, and we describe a method based on HPLC-ICPMS/ESMS to quantitatively measure seven of the major arsenolipids present. Sample preparation involved extraction with DCM/methanol, a cleanup step with silica, and conversion of the (oxo)arsenolipids originally present to thio analogues by brief treatment with H2S. Compared to their oxo analogues, the thioarsenolipids showed much sharper peaks on reversed-phase HPLC, which facilitated their resolution and quantification. The compounds were determined by HPLC-ICPMS and HPLC-ESMS, which provided both arsenic-selective detection and high resolution molecular mass detection of the arsenolipids. In this way, the concentrations of two arsenic-containing hydrocarbons and five arsenosugar phospholipids are reported in the CRM Hijiki. This material may serve as a convenient source of characterized arsenolipids to delineate the presence of these compounds in seafoods and to facilitate research in a new era of arsenic biochemistry.

  11. Determination of polychlorinated biphenyl congeners and chlorinated pesticides in a fish tissue standard reference material.

    PubMed

    Poster, Dianne L; Kucklick, John R; Schantz, Michele M; Porter, Barbara J; Leigh, Stefan D; Wise, Stephen A

    2003-01-01

    The concentrations of a wide range of polychlorinated biphenyl congeners (PCBs) and chlorinated pesticides in a fish tissue Standard Reference Material (SRM) have been determined using multiple methods of analysis. This material, SRM 1946, Lake Superior Fish Tissue, was recently issued by the National Institute of Standards and Technology (NIST) and complements a suite of marine environmental natural-matrix SRMs that are currently available from NIST for the determination of organic contaminants such as aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), PCBs, and chlorinated pesticides. SRM 1946 is a fresh tissue homogenate (frozen) prepared from filleted adult lake trout (Salvelinus namaycush namaycush) collected from the Apostle Islands region of Lake Superior. SRM 1946 has certified and reference concentrations for PCB congeners, including the three non- ortho PCB congeners, and chlorinated pesticides. Certified concentrations are available for 30 PCB congeners and 15 chlorinated pesticides. Reference concentrations are available for 12 PCB congeners and 2 chlorinated pesticides. In addition, SRM 1946 is characterized for additional chemical constituents and properties: fatty acids, extractable fat, methylmercury, total mercury, selected trace elements, proximates, and caloric content. The characterization of chlorinated compounds is described in this paper with an emphasis on the approach used for the certification of the concentrations of PCB congeners and chlorinated pesticides. The PCB congener and chlorinated pesticide data are also compared to concentrations in other marine natural-matrix reference materials available from NIST (fish oil, mussel tissue, whale blubber, and a second fresh frozen fish tissue homogenate prepared from filleted adult lake trout collected from Lake Michigan) and from other organizations such as the National Research Council Canada (ground whole carp), the International Atomic Energy Agency (fish homogenate), and the

  12. Influence of skin tissue properties on the radial reference point for glucose measurement

    NASA Astrophysics Data System (ADS)

    Yang, Yue; Xu, Kexin; Ding, Lijun; Shi, Zhenzhi; Chen, Wenliang

    2009-02-01

    A reference position where the diffuse reflectance light intensity is insensitive to the variation of glucose concentration exists in the radial detection space for glucose measurement in the scattering medium such as skin. The signal measured in this position could be used as an inside reference to evaluate the influence on spectrum caused by other interferential factors. The relationship between the position of radial reference point and the skin tissue property is studied in this paper. Three-layer skin models with different optical parameters are designed to get sample sets at 1200~1700nm. In these sets, μa, μs and g of dermis varies respectively, so does the depth of epidermis or dermis. The distribution rule of dispersion of diffuse reflectance light intensity in the radial space is confirmed with the glucose concentration changes. And the distribution property of the radial reference position in every sample set is obtained through Monte Carlo simulation. The result shows that the distance of radial reference position from light source is insensitive to the variation of absorption coefficient or the depth of dermis, but an increased scattering coefficient will shorten the distance; an increased anisotropy coefficient or depth of epidermis will lengthen it. On the basis of that, the optical probes with different structures are designed according to the skin tissue properties. So they could be used for the measurement of corresponding patients, which enhances the practicability of floating reference method greatly.

  13. The other mechanism of muscular referred pain: the "connective tissue" theory.

    PubMed

    Han, Dong-Gyun

    2009-09-01

    Muscular referred pain, that is, pain perceived in a somatic area other than the site of the noxious stimulation, takes place on a specific place to each muscle in constant and predictable pattern. The central hyperexcitability theory focused on spinal cord, the most proper theory at present, can explain well the segmental pattern of referred pain showing delayed onset. But it is hard to explain the non segmental pattern of referred pain areas of superficial-seated or limb girdle and limb muscles. Referred pain areas of limb girdle and limb muscles appear on the skin above a belt of synergistic muscles beyond the segmental areas. In the case of forearm and calf muscles, referred pain shows up on the palm and sole, the point of force application to the outer object. This finding reflects biomechanical relationship between muscle and its referred pain area. From the phylogenetic perspective, aquatic vertebrated animals (e.g. fish) use myoseptum surrounding myomere, connected to skin to keep tensile strength with it for effective swimming. Likewise, in terrestrial vertebrated animals, there are skin parts weakly interconnected with muscles, though the tensile property of nearly all the skin devolutes except the points of action with the outside. These points are dynamic maximal skin tension areas connected with muscles through superficial fascia, in other words, referred pain areas. Referred pain of deep-seated or truncal muscles appears on the trunk segmentally via spinal cord (the central hyperexcitability theory), but superficial-seated or limb girdle and limb muscles elicit referred pain on dynamic maximal skin tension area through connective tissue (the "connective tissue" theory).

  14. Quantification of Complex Polycyclic Aromatic Hydrocarbon Mixtures in Standard Reference Materials Using GC×GC/ToF-MS

    PubMed Central

    Manzano, Carlos; Hoh, Eunha; Massey Simonich, Staci L.

    2014-01-01

    This research is the first to quantify complex PAH mixtures in NIST SRMs using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/ToF-MS), with and without extract cleanup, and reports previously unidentified PAH isomers in the NIST SRMs. We tested a novel, high orthogonality GC column combination (LC-50×NSP-35), as well as with a commonly used column combination (Rtx-5ms×Rxi-17) for the quantification of a complex mixture of 85 different PAHs, including parent (PAHs), alkyl- (MPAHs), nitro- (NPAHs), oxy- (OPAHs), thio- (SPAHs), bromo- (BrPAHs), and chloro-PAHs (ClPAHs) in extracts from two standard reference materials: NIST SRM1650b (diesel particulate matter), with cleanup and NIST SRM1975 (diesel particulate extract), with and without extract cleanup. The LC-50×NSP-35 column combination resulted in an average absolute percent difference of 33.8%, 62.2% and 30.8% compared to the NIST certified PAH concentrations for NIST SRM1650b, NIST SRM1975 with cleanup and NIST SRM1975 without cleanup, while the Rtx-5ms×Rxi-17 resulted in an absolute percent difference of 38.6%, 67.2% and 79.6% for NIST SRM1650b, NIST SRM1975 with cleanup and NIST SRM1975 without cleanup, respectively. This GC×GC/ToF-MS method increases the number of PAHs detected and quantified in complex environmental extracts using a single chromatographic run. Without clean-up, 7 additional compounds were detected and quantified in NIST SRM1975 using the LC-50×NSP-35 column combination. These results suggest that the use of the LC-50×NSP-35 column combination in GC×GC/ToF-MS not only results in better chromatographic resolution and greater orthogonality for the separation of complex PAH mixtures, but can also be used for the accurate quantification of complex PAH mixtures in environmental extracts without cleanup. PMID:23932031

  15. 3D geometry-based quantification of colocalizations in multichannel 3D microscopy images of human soft tissue tumors.

    PubMed

    Wörz, Stefan; Sander, Petra; Pfannmöller, Martin; Rieker, Ralf J; Joos, Stefan; Mechtersheimer, Gunhild; Boukamp, Petra; Lichter, Peter; Rohr, Karl

    2010-08-01

    We introduce a new model-based approach for automatic quantification of colocalizations in multichannel 3D microscopy images. The approach uses different 3D parametric intensity models in conjunction with a model fitting scheme to localize and quantify subcellular structures with high accuracy. The central idea is to determine colocalizations between different channels based on the estimated geometry of the subcellular structures as well as to differentiate between different types of colocalizations. A statistical analysis was performed to assess the significance of the determined colocalizations. This approach was used to successfully analyze about 500 three-channel 3D microscopy images of human soft tissue tumors and controls.

  16. In-vivo assessment of tissue metabolite levels using 1H MRS and the Electric REference To access In vivo Concentrations (ERETIC) method.

    PubMed

    Heinzer-Schweizer, S; De Zanche, N; Pavan, M; Mens, G; Sturzenegger, U; Henning, A; Boesiger, P

    2010-05-01

    Quantitative values of metabolite concentrations in (1)H magnetic resonance spectroscopy have been obtained using the Electric REference To access In vivo Concentrations (ERETIC) method, whereby a synthetic reference signal is injected during the acquisition of spectra. The method has been improved to enable quantification of metabolite concentrations in vivo. Optical signal transmission was used to eliminate random fluctuations in ERETIC signal coupling to the receiver coil due to changes in position of cables and highly dielectric human tissue. Stability and reliability of the signal were tested in vitro, achieving stability with a mean error of 2.83%. Scaling of the signal in variable loading conditions was demonstrated and in-vivo measurements of brain were acquired on a 3T Philips system using a transmit/receive coil. The quantitative brain water and metabolite concentration values are in good agreement with those in the literature.

  17. Nuquantus: Machine learning software for the characterization and quantification of cell nuclei in complex immunofluorescent tissue images

    PubMed Central

    Gross, Polina; Honnorat, Nicolas; Varol, Erdem; Wallner, Markus; Trappanese, Danielle M.; Sharp, Thomas E.; Starosta, Timothy; Duran, Jason M.; Koller, Sarah; Davatzikos, Christos; Houser, Steven R.

    2016-01-01

    Determination of fundamental mechanisms of disease often hinges on histopathology visualization and quantitative image analysis. Currently, the analysis of multi-channel fluorescence tissue images is primarily achieved by manual measurements of tissue cellular content and sub-cellular compartments. Since the current manual methodology for image analysis is a tedious and subjective approach, there is clearly a need for an automated analytical technique to process large-scale image datasets. Here, we introduce Nuquantus (Nuclei quantification utility software) - a novel machine learning-based analytical method, which identifies, quantifies and classifies nuclei based on cells of interest in composite fluorescent tissue images, in which cell borders are not visible. Nuquantus is an adaptive framework that learns the morphological attributes of intact tissue in the presence of anatomical variability and pathological processes. Nuquantus allowed us to robustly perform quantitative image analysis on remodeling cardiac tissue after myocardial infarction. Nuquantus reliably classifies cardiomyocyte versus non-cardiomyocyte nuclei and detects cell proliferation, as well as cell death in different cell classes. Broadly, Nuquantus provides innovative computerized methodology to analyze complex tissue images that significantly facilitates image analysis and minimizes human bias. PMID:27005843

  18. Nuquantus: Machine learning software for the characterization and quantification of cell nuclei in complex immunofluorescent tissue images.

    PubMed

    Gross, Polina; Honnorat, Nicolas; Varol, Erdem; Wallner, Markus; Trappanese, Danielle M; Sharp, Thomas E; Starosta, Timothy; Duran, Jason M; Koller, Sarah; Davatzikos, Christos; Houser, Steven R

    2016-03-23

    Determination of fundamental mechanisms of disease often hinges on histopathology visualization and quantitative image analysis. Currently, the analysis of multi-channel fluorescence tissue images is primarily achieved by manual measurements of tissue cellular content and sub-cellular compartments. Since the current manual methodology for image analysis is a tedious and subjective approach, there is clearly a need for an automated analytical technique to process large-scale image datasets. Here, we introduce Nuquantus (Nuclei quantification utility software) - a novel machine learning-based analytical method, which identifies, quantifies and classifies nuclei based on cells of interest in composite fluorescent tissue images, in which cell borders are not visible. Nuquantus is an adaptive framework that learns the morphological attributes of intact tissue in the presence of anatomical variability and pathological processes. Nuquantus allowed us to robustly perform quantitative image analysis on remodeling cardiac tissue after myocardial infarction. Nuquantus reliably classifies cardiomyocyte versus non-cardiomyocyte nuclei and detects cell proliferation, as well as cell death in different cell classes. Broadly, Nuquantus provides innovative computerized methodology to analyze complex tissue images that significantly facilitates image analysis and minimizes human bias.

  19. Nuquantus: Machine learning software for the characterization and quantification of cell nuclei in complex immunofluorescent tissue images

    NASA Astrophysics Data System (ADS)

    Gross, Polina; Honnorat, Nicolas; Varol, Erdem; Wallner, Markus; Trappanese, Danielle M.; Sharp, Thomas E.; Starosta, Timothy; Duran, Jason M.; Koller, Sarah; Davatzikos, Christos; Houser, Steven R.

    2016-03-01

    Determination of fundamental mechanisms of disease often hinges on histopathology visualization and quantitative image analysis. Currently, the analysis of multi-channel fluorescence tissue images is primarily achieved by manual measurements of tissue cellular content and sub-cellular compartments. Since the current manual methodology for image analysis is a tedious and subjective approach, there is clearly a need for an automated analytical technique to process large-scale image datasets. Here, we introduce Nuquantus (Nuclei quantification utility software) - a novel machine learning-based analytical method, which identifies, quantifies and classifies nuclei based on cells of interest in composite fluorescent tissue images, in which cell borders are not visible. Nuquantus is an adaptive framework that learns the morphological attributes of intact tissue in the presence of anatomical variability and pathological processes. Nuquantus allowed us to robustly perform quantitative image analysis on remodeling cardiac tissue after myocardial infarction. Nuquantus reliably classifies cardiomyocyte versus non-cardiomyocyte nuclei and detects cell proliferation, as well as cell death in different cell classes. Broadly, Nuquantus provides innovative computerized methodology to analyze complex tissue images that significantly facilitates image analysis and minimizes human bias.

  20. Evaluation of reference genes for gene expression studies in human brown adipose tissue.

    PubMed

    Taube, Magdalena; Andersson-Assarsson, Johanna C; Lindberg, Kristin; Pereira, Maria J; Gäbel, Markus; Svensson, Maria K; Eriksson, Jan W; Svensson, Per-Arne

    2015-01-01

    Human brown adipose tissue (BAT) has during the last 5 year been subjected to an increasing research interest, due to its putative function as a target for future obesity treatments. The most commonly used method for molecular studies of human BAT is the quantitative polymerase chain reaction (qPCR). This method requires normalization to a reference gene (genes with uniform expression under different experimental conditions, e.g. similar expression levels between human BAT and WAT), but so far no evaluation of reference genes for human BAT has been performed. Two different microarray datasets with samples containing human BAT were used to search for genes with low variability in expression levels. Seven genes (FAM96B, GNB1, GNB2, HUWE1, PSMB2, RING1 and TPT1) identified by microarray analysis, and 8 commonly used reference genes (18S, B2M, GAPDH, LRP10, PPIA, RPLP0, UBC, and YWHAZ) were selected and further analyzed by quantitative PCR in both BAT containing perirenal adipose tissue and subcutaneous adipose tissue. Results were analyzed using 2 different algorithms (Normfinder and geNorm). Most of the commonly used reference genes displayed acceptably low variability (geNorm M-values <0.5) in the samples analyzed, but the novel reference genes identified by microarray displayed an even lower variability (M-values <0.25). Our data suggests that PSMB2, GNB2 and GNB1 are suitable novel reference genes for qPCR analysis of human BAT and we recommend that they are included in future gene expression studies of human BAT.

  1. Spatially resolved quantification of gadolinium(III)-based magnetic resonance agents in tissue by MALDI imaging mass spectrometry after in vivo MRI.

    PubMed

    Aichler, Michaela; Huber, Katharina; Schilling, Franz; Lohöfer, Fabian; Kosanke, Katja; Meier, Reinhard; Rummeny, Ernst J; Walch, Axel; Wildgruber, Moritz

    2015-03-27

    Gadolinium(III)-based contrast agents improve the sensitivity and specificity of magnetic resonance imaging (MRI), especially when targeted contrast agents are applied. Because of nonlinear correlation between the contrast agent concentration in tissue and the MRI signal obtained in vivo, quantification of certain biological or pathophysiological processes by MRI remains a challenge. Up to now, no technology has been able to provide a spatially resolved quantification of MRI agents directly within the tissue, which would allow a more precise verification of in vivo imaging results. MALDI imaging mass spectrometry for spatially resolved in situ quantification of gadolinium(III) agents, in correlation to in vivo MRI, were evaluated. Enhanced kinetics of Gadofluorine M were determined dynamically over time in a mouse model of myocardial infarction. MALDI imaging was able to corroborate the in vivo imaging MRI signals and enabled in situ quantification of the gadolinium probe with high spatial resolution.

  2. Multivariate reference technique for quantitative analysis of fiber-optic tissue Raman spectroscopy.

    PubMed

    Bergholt, Mads Sylvest; Duraipandian, Shiyamala; Zheng, Wei; Huang, Zhiwei

    2013-12-03

    We report a novel method making use of multivariate reference signals of fused silica and sapphire Raman signals generated from a ball-lens fiber-optic Raman probe for quantitative analysis of in vivo tissue Raman measurements in real time. Partial least-squares (PLS) regression modeling is applied to extract the characteristic internal reference Raman signals (e.g., shoulder of the prominent fused silica boson peak (~130 cm(-1)); distinct sapphire ball-lens peaks (380, 417, 646, and 751 cm(-1))) from the ball-lens fiber-optic Raman probe for quantitative analysis of fiber-optic Raman spectroscopy. To evaluate the analytical value of this novel multivariate reference technique, a rapid Raman spectroscopy system coupled with a ball-lens fiber-optic Raman probe is used for in vivo oral tissue Raman measurements (n = 25 subjects) under 785 nm laser excitation powers ranging from 5 to 65 mW. An accurate linear relationship (R(2) = 0.981) with a root-mean-square error of cross validation (RMSECV) of 2.5 mW can be obtained for predicting the laser excitation power changes based on a leave-one-subject-out cross-validation, which is superior to the normal univariate reference method (RMSE = 6.2 mW). A root-mean-square error of prediction (RMSEP) of 2.4 mW (R(2) = 0.985) can also be achieved for laser power prediction in real time when we applied the multivariate method independently on the five new subjects (n = 166 spectra). We further apply the multivariate reference technique for quantitative analysis of gelatin tissue phantoms that gives rise to an RMSEP of ~2.0% (R(2) = 0.998) independent of laser excitation power variations. This work demonstrates that multivariate reference technique can be advantageously used to monitor and correct the variations of laser excitation power and fiber coupling efficiency in situ for standardizing the tissue Raman intensity to realize quantitative analysis of tissue Raman measurements in vivo, which is particularly appealing in

  3. Reference tissue modeling with parameter coupling: application to a study of SERT binding in HIV

    NASA Astrophysics Data System (ADS)

    Endres, Christopher J.; Hammoud, Dima A.; Pomper, Martin G.

    2011-04-01

    When applicable, it is generally preferred to evaluate positron emission tomography (PET) studies using a reference tissue-based approach as that avoids the need for invasive arterial blood sampling. However, most reference tissue methods have been shown to have a bias that is dependent on the level of tracer binding, and the variability of parameter estimates may be substantially affected by noise level. In a study of serotonin transporter (SERT) binding in HIV dementia, it was determined that applying parameter coupling to the simplified reference tissue model (SRTM) reduced the variability of parameter estimates and yielded the strongest between-group significant differences in SERT binding. The use of parameter coupling makes the application of SRTM more consistent with conventional blood input models and reduces the total number of fitted parameters, thus should yield more robust parameter estimates. Here, we provide a detailed evaluation of the application of parameter constraint and parameter coupling to [11C]DASB PET studies. Five quantitative methods, including three methods that constrain the reference tissue clearance (kr2) to a common value across regions were applied to the clinical and simulated data to compare measurement of the tracer binding potential (BPND). Compared with standard SRTM, either coupling of kr2 across regions or constraining kr2 to a first-pass estimate improved the sensitivity of SRTM to measuring a significant difference in BPND between patients and controls. Parameter coupling was particularly effective in reducing the variance of parameter estimates, which was less than 50% of the variance obtained with standard SRTM. A linear approach was also improved when constraining kr2 to a first-pass estimate, although the SRTM-based methods yielded stronger significant differences when applied to the clinical study. This work shows that parameter coupling reduces the variance of parameter estimates and may better discriminate between

  4. Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay.

    PubMed

    Reeves, R Keith; Evans, Tristan I; Gillis, Jacqueline; Wong, Fay E; Connole, Michelle; Carville, Angela; Johnson, R Paul

    2011-03-31

    Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.

  5. A specific endogenous reference for genetically modified common bean (Phaseolus vulgaris L.) DNA quantification by real-time PCR targeting lectin gene.

    PubMed

    Venturelli, Gustavo L; Brod, Fábio C A; Rossi, Gabriela B; Zimmermann, Naíra F; Oliveira, Jaison P; Faria, Josias C; Arisi, Ana C M

    2014-11-01

    The Embrapa 5.1 genetically modified (GM) common bean was approved for commercialization in Brazil. Methods for the quantification of this new genetically modified organism (GMO) are necessary. The development of a suitable endogenous reference is essential for GMO quantification by real-time PCR. Based on this, a new taxon-specific endogenous reference quantification assay was developed for Phaseolus vulgaris L. Three genes encoding common bean proteins (phaseolin, arcelin, and lectin) were selected as candidates for endogenous reference. Primers targeting these candidate genes were designed and the detection was evaluated using the SYBR Green chemistry. The assay targeting lectin gene showed higher specificity than the remaining assays, and a hydrolysis probe was then designed. This assay showed high specificity for 50 common bean samples from two gene pools, Andean and Mesoamerican. For GM common bean varieties, the results were similar to those obtained for non-GM isogenic varieties with PCR efficiency values ranging from 92 to 101 %. Moreover, this assay presented a limit of detection of ten haploid genome copies. The primers and probe developed in this work are suitable to detect and quantify either GM or non-GM common bean.

  6. Origin and quantification of differences between normal and tumor tissues observed by terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Sayuri; Fukushi, Yasuko; Kubota, Oichi; Itsuji, Takeaki; Ouchi, Toshihiko; Yamamoto, Seiji

    2016-09-01

    The origin of the differences in the refractive index observed between normal and tumor tissues using terahertz spectroscopy has been described quantitatively. To estimate water content differences in tissues, we prepared fresh and paraffin-embedded samples from rats. An approximately 5% increase of water content in tumor tissues was calculated from terahertz time domain spectroscopy measurements compared to normal tissues. A greater than 15% increase in percentage of cell nuclei per unit area in tumor tissues was observed by hematoxylin and eosin stained samples, which generates a higher refractive index of biological components other than water. Both high water content and high cell density resulted in higher refractive index by approximately 0.05 in tumor tissues. It is predicted that terahertz spectroscopy can also be used to detect brain tumors in human tissue due to the same underlying mechanism as in rats.

  7. Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues*

    PubMed Central

    Steiner, Carine; Tille, Jean-Christophe; Lamerz, Jens; Kux van Geijtenbeek, Sabine; McKee, Thomas A.; Venturi, Miro; Rubbia-Brandt, Laura; Hochstrasser, Denis; Cutler, Paul; Lescuyer, Pierre; Ducret, Axel

    2015-01-01

    The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R2: 0.99–1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry

  8. An image-based skeletal tissue model for the ICRP reference newborn

    NASA Astrophysics Data System (ADS)

    Pafundi, Deanna; Lee, Choonsik; Watchman, Christopher; Bourke, Vincent; Aris, John; Shagina, Natalia; Harrison, John; Fell, Tim; Bolch, Wesley

    2009-07-01

    Hybrid phantoms represent a third generation of computational models of human anatomy needed for dose assessment in both external and internal radiation exposures. Recently, we presented the first whole-body hybrid phantom of the ICRP reference newborn with a skeleton constructed from both non-uniform rational B-spline and polygon-mesh surfaces (Lee et al 2007 Phys. Med. Biol. 52 3309-33). The skeleton in that model included regions of cartilage and fibrous connective tissue, with the remainder given as a homogenous mixture of cortical and trabecular bone, active marrow and miscellaneous skeletal tissues. In the present study, we present a comprehensive skeletal tissue model of the ICRP reference newborn to permit a heterogeneous representation of the skeleton in that hybrid phantom set—both male and female—that explicitly includes a delineation of cortical bone so that marrow shielding effects are correctly modeled for low-energy photons incident upon the newborn skeleton. Data sources for the tissue model were threefold. First, skeletal site-dependent volumes of homogeneous bone were obtained from whole-cadaver CT image analyses. Second, selected newborn bone specimens were acquired at autopsy and subjected to micro-CT image analysis to derive model parameters of the marrow cavity and bone trabecular 3D microarchitecture. Third, data given in ICRP Publications 70 and 89 were selected to match reference values on total skeletal tissue mass. Active marrow distributions were found to be in reasonable agreement with those given previously by the ICRP. However, significant differences were seen in total skeletal and site-specific masses of trabecular and cortical bone between the current and ICRP newborn skeletal tissue models. The latter utilizes an age-independent ratio of 80%/20% cortical and trabecular bone for the reference newborn. In the current study, a ratio closer to 40%/60% is used based upon newborn CT and micro-CT skeletal image analyses. These

  9. Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing

    PubMed Central

    Hoang, Margaret L.; Kinde, Isaac; Tomasetti, Cristian; McMahon, K. Wyatt; Rosenquist, Thomas A.; Grollman, Arthur P.; Kinzler, Kenneth W.; Vogelstein, Bert; Papadopoulos, Nickolas

    2016-01-01

    We present the bottleneck sequencing system (BotSeqS), a next-generation sequencing method that simultaneously quantifies rare somatic point mutations across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately before library amplification. We use BotSeqS to show age- and tissue-dependent accumulations of rare mutations and demonstrate that somatic mutational burden in normal human tissues can vary by several orders of magnitude, depending on biologic and environmental factors. We further show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, the mutation spectra of normal tissues were different from each other, but similar to those of the cancers that arose in them. This technology can provide insights into the number and nature of genetic alterations in normal tissues and can be used to address a variety of fundamental questions about the genomes of diseased tissues. PMID:27528664

  10. Reliability of soft tissue references for anteroposterior measurement of dental bases.

    PubMed

    Ferrario, V F; Sforza, C; Serrao, G; Colombo, A; Ciusa, V; Bignotto, M

    1998-01-01

    The aims of the present investigation were to devise a new anteroposterior measurement of maxillomandibular discrepancies that would consider both hard and soft tissue contributions, and to verify the correlation of this measurement to a well-established linear assessment of anteroposterior discrepancy. On the pretreatment lateral cephalographs of 300 orthodontic patients (162 males, 138 females) aged between 6 and 50 years, the Wits appraisal and a new "soft tissue" Wits appraisal (linear distance between the projections of soft tissue A and B points on the bisecting occlusal plane) were measured. In the analyzed sample, the former was more variable than the latter. The two measurements were significantly correlated to each other without sex- or age-characteristic patterns. From the correlation, reference values for the new measurement were estimated and found to be between -1.9 mm and 5.4 mm for individuals with a normal occlusion. The new measurement could allow a more careful evaluation of the soft tissue drape together with the underlying hard tissue structure.

  11. Quantification of nucleic acid quality in postmortem tissues from a cancer research autopsy program

    PubMed Central

    Fan, Jun; Khanin, Raya; Sakamoto, Hitomi; Zhong, Yi; Michael, Chelsea; Pena, Derwin; Javier, Breanna; Wood, Laura D.; Iacobuzio-Donahue, Christine A.

    2016-01-01

    The last decade has seen a marked rise in the use of cancer tissues obtained from research autopsies. Such resources have been invaluable for studying cancer evolution or the mechanisms of therapeutic resistance to targeted therapies. Degradation of biomolecules is a potential challenge to usage of cancer tissues obtained in the post-mortem setting and remains incompletely studied. We analysed the nucleic acid quality in 371 different frozen tissue samples collected from 80 patients who underwent a research autopsy, including eight normal tissue types, primary and metastatic tumors. Our results indicate that RNA integrity number (RIN) of normal tissues decline with the elongation of post-mortem interval (PMI) in a tissue-type specific manner. Unlike normal tissues, the RNA quality of cancer tissues is highly variable with respect to post-mortem interval. The kinetics of DNA damage also has tissue type-specific features. Moreover, while DNA degradation is an indicator of low RNA quality, the converse is not true. Finally, we show that despite RIN values as low as 5.0, robust data can be obtained by RNA sequencing that reliably discriminates expression signatures. PMID:27602498

  12. A microsatellite based method for quantification of fungi in decomposing plant material elucidates the role of Fusarium graminearum DON production in the saprophytic competition with Trichoderma atroviride in maize tissue microcosms.

    PubMed

    Naef, Andreas; Senatore, Mauro; Défago, Geneviève

    2006-02-01

    Common PCR assays for quantification of fungi in living plants cannot be used to study saprophytic colonization of fungi because plant decomposition releases PCR-inhibiting substances and saprophytes degrade the plant DNA which could serve as internal standard. The microsatellite PCR assays presented here overcome these problems by spiking samples prior to DNA extraction with mycelium of a reference strain. PCR with fluorescent primers co-amplifies microsatellite fragments of different length from target and reference strains. These fragments were separated in a capillary sequencer with fluorescence detection. The target/reference ratio of fluorescence signal was used to calculate target biomass in the sample. Such PCR assays were developed for the mycotoxin deoxynivalenol (DON)-producing wheat and maize pathogen Fusarium graminearum and the biocontrol agent Trichoderma atroviride, using new microsatellite markers. In contrast to real-time PCR assays, the novel PCR assays showed reliable fungal biomass quantification in samples with differentially decomposed plant tissue. The PCR assays were used to quantify the two fungi after competitive colonization of autoclaved maize leaf tissue in microcosms. Using a DON-producing F. graminearum wild-type strain and its nontoxigenic mutant we found no evidence for a role of DON production in F. graminearum defense against T. atroviride. The presence of T. atroviride resulted in a 36% lower wild-type DON production per biomass.

  13. A mussel tissue certified reference material for multiple phycotoxins. Part 1: design and preparation.

    PubMed

    McCarron, Pearse; Emteborg, Håkan; Nulty, Cíara; Rundberget, Thomas; Loader, Jared I; Teipel, Katharina; Miles, Christopher O; Quilliam, Michael A; Hess, Philipp

    2011-05-01

    The development of multi-analyte methods for lipophilic shellfish toxins based on liquid chromatography-mass spectrometry permits rapid screening and analysis of samples for a wide variety of toxins in a single run. Validated methods and appropriate certified reference materials (CRMs) are required to ensure accuracy of results. CRMs are essential for accurate instrument calibration, for assessing the complete analytical method from sample extraction to data analysis and for verifying trueness. However, CRMs have hitherto only been available for single toxin groups. Production of a CRM containing six major toxin groups was achieved through an international collaboration. Preparation of this material, CRM-FDMT1, drew on information from earlier studies as well as improved methods for isolation of toxins, handling bulk tissues and production of reference materials. Previous investigations of stabilisation techniques indicated freeze-drying to be a suitable procedure for preparation of shellfish toxin reference materials and applicable to a wide range of toxins. CRM-FDMT1 was initially prepared as a bulk wet tissue homogenate containing domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxin-2, yessotoxin and 13-desmethylspirolide C. The homogenate was then freeze-dried, milled and bottled in aliquots suitable for distribution and analysis. The moisture content and particle size distribution were measured, and determined to be appropriate. A preliminary toxin analysis of the final material showed a comprehensive toxin profile.

  14. Accuracy of dual-energy computed tomography for the quantification of iodine in a soft tissue-mimicking phantom.

    PubMed

    Li, Jung-Hui; Du, Yeh-Ming; Huang, Hsuan-Ming

    2015-09-08

    The objective of this study was to evaluate the accuracy of dual-energy CT (DECT) for quantifying iodine using a soft tissue-mimicking phantom across various DECT acquisition parameters and dual-source CT (DSCT) scanners. A phantom was constructed with plastic tubes containing soft tissue-mimicking materials with known iodine concentrations (0-20 mg/mL). Experiments were performed on two DSCT scanners, one equipped with an integrated detector and the other with a conventional detector. DECT data were acquired using two DE modes (80 kV/Sn140 kV and 100 kV/Sn140 kV) with four pitch values (0.6, 0.8, 1.0, and 1.2). Images were reconstructed using a soft tissue kernel with and without beam hardening correction (BHC) for iodine. Using the dedicated DE software, iodine concentrations were measured and compared to true concentrations. We also investigated the effect of reducing gantry rotation time on the DECT-based iodine measurement. At iodine concentrations higher than 10 mg/mL, the relative error in measured iodine concentration increased slightly. This error can be decreased by using the kernel with BHC, compared with the kernel without BHC. Both 80 kV/Sn140 kV and 100 kV/Sn140 kV modes could provide accurate quantification of iodine content. Increasing pitch value or reducing gantry rotation time had only a minor impact on the DECT-based iodine measurement. The DSCT scanner, equipped with the new integrated detector, showed more accurate iodine quantification for all iodine concentrations higher than 10 mg/mL. An accurate quantification of iodine can be obtained using the second-generation DSCT scanner in various DE modes with pitch values up to 1.2 and gantry rotation time down to 0.28 s. For iodine concentrations ≥ 10 mg/mL, using the new integrated detector and the kernel with BHC can improve the accuracy of DECT-based iodine measurements.

  15. Accuracy of dual-energy computed tomography for the quantification of iodine in a soft tissue-mimicking phantom.

    PubMed

    Li, Jung-Hui; Du, Yeh-Ming; Huang, Hsuan-Ming

    2015-09-01

    The objective of this study was to evaluate the accuracy of dual-energy CT (DECT) for quantifying iodine using a soft tissue-mimicking phantom across various DECT acquisition parameters and dual-source CT (DSCT) scanners. A phantom was constructed with plastic tubes containing soft tissue-mimicking materials with known iodine concentrations (0-20 mg/mL). Experiments were performed on two DSCT scanners, one equipped with an integrated detector and the other with a conventional detector. DECT data were acquired using two DE modes (80 kV/Sn140 kV and 100 kV/Sn140 kV) with four pitch values (0.6, 0.8, 1.0, and 1.2). Images were reconstructed using a soft tissue kernel with and without beam hardening correction (BHC) for iodine. Using the dedicated DE software, iodine concentrations were measured and compared to true concentrations. We also investigated the effect of reducing gantry rotation time on the DECT-based iodine measurement. At iodine concentrations higher than 10 mg/mL, the relative error in measured iodine concentration increased slightly. This error can be decreased by using the kernel with BHC, compared with the kernel without BHC. Both 80 kV/Sn140 kV and 100 kV/Sn140 kV modes could provide accurate quantification of iodine content. Increasing pitch value or reducing gantry rotation time had only a minor impact on the DECT-based iodine measurement. The DSCT scanner, equipped with the new integrated detector, showed more accurate iodine quantification for all iodine concentrations higher than 10 mg/mL. An accurate quantification of iodine can be obtained using the second-generation DSCT scanner in various DE modes with pitch values up to 1.2 and gantry rotation time down to 0.28 s. For iodine concentrations ≥10 mg/mL, using the new integrated detector and the kernel with BHC can improve the accuracy of DECT-based iodine measurements. PACS number: 87.57.Q.

  16. Magnetic Nanoparticle Location and Quantification in Mice Tissues after Intravenous Injection

    NASA Astrophysics Data System (ADS)

    Gutiérrez, Lucía; Cabrera, Lourdes I.; Mejías, Raquel; Barber, Domingo F.; Serna, Carlos J.; Morales, M. Puerto

    2010-10-01

    DMSA-coated magnetic nanoparticles have been used for drug delivery, in particular to transport cytokines towards an induced tumour in a murine model. In this work, the use of transmission electron microscopy and AC magnetic susceptibility measurements of the tissue have allowed the detection of the particles within the target tissue.

  17. Optimization and evaluation of MALDI TOF mass spectrometric imaging for quantification of orally dosed octreotide in mouse tissues.

    PubMed

    Rao, Tai; Shen, Boyu; Zhu, Zhangpei; Shao, Yuhao; Kang, Dian; Li, Xinuo; Yin, Xiaoxi; Li, Haofeng; Xie, Lin; Wang, Guangji; Liang, Yan

    2017-04-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI) has received considerable attention in recent years since it allows molecular mapping of diverse bimolecular in animal/plant tissue sections, although some barriers still exist in absolute pixel-to-pixel quantification. Octreotide, a synthetic somatostatin analogue, has been widely used to prevent gastrointestine bleeding in the clinic. The aim of the present study is to develop a MALDI-TOF-MSI method for quantitatively visualizing spatial distribution of octreotide in mouse tissues. In this process, a structurally similar internal standard was spotted onto tissue section together with matrix solution to minimize signal variation and give excellent quantitative results. The 2,5-dihydroxybenzoic acid was chosen as the most suitable matrix via comparing the signal/noise generated by MALDI-TOF-MSI after cocrystallization of octreotide with different matrix candidates. The reliability of MALDI-TOF-MSI, with respect to linearity, sensitivity and precision, was tested via measuring octreotide in fresh tissue slices at different concentrations. The validated method was then successfully applied to visualize the distribution of octreotide in mouse tissues after oral administration of octreotide at 20mg/kg. The results demonstrated that MALDI-TOF-MSI could not only clearly visualize the spatial distribution of octreotide, but also make the calculation of the key pharmacokinetic parameters (Tmax and t1/2) possible. More importantly, the tissue concentration-time curves of octreotide determined by MALDI-TOF-MSI agreed well with those measured based on LC-MS/MS.These findings illustrate the potential of MALDI-TOF-MSI in pharmacokinetic profiling during drug development.

  18. Simultaneous identification and quantification of new psychoactive substances in blood by GC-APCI-QTOFMS coupled to nitrogen chemiluminescence detection without authentic reference standards.

    PubMed

    Ojanperä, Ilkka; Mesihää, Samuel; Rasanen, Ilpo; Pelander, Anna; Ketola, Raimo A

    2016-05-01

    A novel platform is introduced for simultaneous identification and quantification of new psychoactive substances (NPS) in blood matrix, without the necessity of using authentic reference standards. The instrumentation consisted of gas chromatography (GC) coupled to nitrogen chemiluminescence detection (NCD) and atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometry (APCI-QTOFMS). In this concept, the GC flow is divided in appropriate proportions between NCD for single-calibrant quantification, utilizing the detector's equimolar response to nitrogen, and QTOFMS for accurate mass-based identification. The principle was proven by analyzing five NPS, bupropion, desoxypipradrol (2-DPMP), mephedrone, methylone, and naphyrone, in sheep blood. The samples were spiked with the analytes post-extraction to avoid recovery considerations at this point. All the NPS studies produced a protonated molecule in APCI resulting in predictable fragmentation with high mass accuracy. The N-equimolarity of quantification by NCD was investigated by using external calibration with the secondary standard caffeine at five concentration levels between 0.17 and 1.7 mg/L in blood matrix as five replicates. The equimolarity was on average 98.7%, and the range of individual equimolarity determinations was 76.7-130.1%. The current analysis platform affords a promising approach to instant simultaneous qualitative and quantitative analysis of drugs in the absence of authentic reference standards, not only in forensic and clinical toxicology but also in other bioanalytical applications.

  19. Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR

    PubMed Central

    2014-01-01

    Background Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed. Results We describe a qPCR technique based on the single copy gene β’ DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes. Conclusions This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak. PMID:24767577

  20. Quantification of Regional Interstitial Lung Disease from CT-derived Fractional Tissue Volume: A Lung Tissue Research Consortium Study

    PubMed Central

    Yilmaz, Cuneyt; Watharkar, Snehal S.; de Leon, Alberto Diaz; Garcia, Christine K.; Patel, Nova C.; Jordan, Kirk G.; Hsia, Connie C.W.

    2011-01-01

    Rationale and Objectives Evaluation of chest CT is usually qualitative or semi-quantitative, resulting in subjective descriptions often by different observers over time and imprecise determinations of disease severity within distorted lobes. There is a need for standardized imaging biomarkers to quantify regional disease, maximize diagnostic yield, and facilitate multi-center comparisons. We applied lobe-based voxelwise image analysis to derive regional air (Vair) and tissue (Vtissue) volumes and fractional tissue volume (FTV=tissue/[tissue+air] volume) as internally standardized parameter for assessing interstitial lung disease (ILD). Materials and Methods High-resolution CT was obtained at supine and prone end-inspiration and supine end-expiration in 29 patients with ILD and 20 normal subjects. Lobar Vair, Vtissue, and FTV were expressed along standard coordinate axes. Results In normal subjects from end-inspiration to end-expiration, total Vair declined 43%, FTV increased ~80% while Vtissue remained unchanged. With increasing ILD, Vair declined and Vtissue rose in all lobes; FTV increased with a peripheral-to-central progression inversely correlated to spirometry and lung diffusing capacity (R2=0.57–0.75, prone end-inspiration). Inter- and intra-lobar coefficients of variation (CVs) of FTV increased 84–148% in mild-to-moderate ILD, indicating greater spatial heterogeneity, then normalized in severe ILD. Analysis of discontinuous images incurs <3% error compared to consecutive images. Conclusions These regional attenuation-based biomarkers could quantify heterogeneous parenchymal disease in distorted lobes, detect mild ILD involvement in all lobes and describe the pattern of disease progression. The next step would be to study a larger series, examine reproducibility and follow longitudinal changes in correlation with clinical and functional indices. PMID:21596593

  1. High-performance capillary electrophoretic method for the quantification of 5-methyl 2'-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues.

    PubMed

    Fraga, Mario F; Uriol, Esther; Borja Diego, L; Berdasco, María; Esteller, Manel; Cañal, María Jesús; Rodríguez, Roberto

    2002-06-01

    A new approach to the evaluation of the relative degree of genomic DNA methylation through the quantification of 2'-deoxynucleosides is proposed. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA has been performed using micellar high-performance capillary electrophoresis (HPCE) with UV-Vis detection. This approach has been demonstrated to be more sensitive and specific than other HPCE methods for the quantification of DNA methylation degree and also to be faster than other HPLC-based methods. The detection and quantification of nucleosides through enzymatic hydrolyses notably increases the specificity of the technique and allows its exploitation in the analysis of poorly purified and/or concentrated DNA samples such as those obtained from meristematic plant regions and paraffin-embedded tissues.

  2. New biological reference materials - in vivo incorporated toxic metals in water hyacinth tissues

    SciTech Connect

    Austin, J.R.; Simon, S.J.; Williams, L.R.; Beckert, W.F.

    1985-06-01

    The purpose of this study was to demonstrate that high-quality reference materials, containing high levels of multiple toxic elements, can be produced with in vivo incorporation procedures. The approach taken was to produce water hyacinth tissue materials - leaves and stems containing high levels of arsenic, cadmium, lead, and mercury - as follows: apply a hydroponic feeding procedure for the in vivo incorporation of toxic elements into water hyacinths; dry, blend, and homogenize the plant materials and determine the levels of the incorporated elements and the homogeneity of the generated plant material; demonstrate that low-level control materials can be successfully blended with high-level materials to yield a homogeneous material with intermediate toxicant levels; evaluate the precision of the analytical methods used to determine toxic element levels in the materials; and evaluate the stability of the resulting materials. Sufficient quantities of the parent materials were produced so that characterized reference materials can now be made available on request. Levels of the toxic elements incorporated in water hyacinth leaves were 100, 300, 60, and 27 times the levels present in the control leaves for arsenic, cadmium, lead, and mercury, respectively. Overall precision of sampling, subsampling, and digestion, and chemical analysis of the treated materials, ranged from 3 to 10% relative standard deviation and was generally comparable to that of three NBS biological reference materials tested. 3 references, 1 figure, 4 tables.

  3. Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering*

    PubMed Central

    Hill, Ryan C.; Calle, Elizabeth A.; Dzieciatkowska, Monika; Niklason, Laura E.; Hansen, Kirk C.

    2015-01-01

    The use of extracellular matrix (ECM)1 scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs. PMID:25660013

  4. A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in tumor and tissues.

    PubMed

    Lucas, Andrew T; O'Neal, Sara K; Santos, Charlene M; White, Taylor F; Zamboni, William C

    2016-02-05

    Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of the intracellular active form of doxorubicin. Thus, the development of a sample processing method and a high-performance liquid chromatography (HPLC) methodology was performed in order to quantify doxorubicin that is associated with DNA in tumors and tissues, which provided an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid-liquid phase extraction to isolate drug associated with DNA; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2μm, 2.0×100mm) analytical column and a gradient mobile phase of 0.1% formic acid in water or acetonitrile for separation and quantification. The assay has a lower limit of detection (LLOQ) of 10ng/mL and is shown to be linear up to 3000ng/mL. The intra- and inter-day precision of the assay expressed as a coefficient of variation (CV%) ranged from 4.01 to 8.81%. Furthermore, the suitability of this assay for measuring doxorubicin associated with DNA in vivo was demonstrated by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72h after administration of PEGylated liposomal doxorubicin (Doxil(®); PLD) at 6mg/kg IV x 1. This HPLC assay allows for sensitive intracellular quantification of doxorubicin and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle formulations of doxorubicin.

  5. Image-based quantification of fiber alignment within electrospun tissue engineering scaffolds is related to mechanical anisotropy.

    PubMed

    Fee, Timothy; Downs, Crawford; Eberhardt, Alan; Zhou, Yong; Berry, Joel

    2016-07-01

    It is well documented that electrospun tissue engineering scaffolds can be fabricated with variable degrees of fiber alignment to produce scaffolds with anisotropic mechanical properties. Several attempts have been made to quantify the degree of fiber alignment within an electrospun scaffold using image-based methods. However, these methods are limited by the inability to produce a quantitative measure of alignment that can be used to make comparisons across publications. Therefore, we have developed a new approach to quantifying the alignment present within a scaffold from scanning electron microscopic (SEM) images. The alignment is determined by using the Sobel approximation of the image gradient to determine the distribution of gradient angles with an image. This data was fit to a Von Mises distribution to find the dispersion parameter κ, which was used as a quantitative measure of fiber alignment. We fabricated four groups of electrospun polycaprolactone (PCL) + Gelatin scaffolds with alignments ranging from κ = 1.9 (aligned) to κ = 0.25 (random) and tested our alignment quantification method on these scaffolds. It was found that our alignment quantification method could distinguish between scaffolds of different alignments more accurately than two other published methods. Additionally, the alignment parameter κ was found to be a good predictor the mechanical anisotropy of our electrospun scaffolds. The ability to quantify fiber alignment within and make direct comparisons of scaffold fiber alignment across publications can reduce ambiguity between published results where cells are cultured on "highly aligned" fibrous scaffolds. This could have important implications for characterizing mechanics and cellular behavior on aligned tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1680-1686, 2016.

  6. Time domain diffuse optical spectroscopy: In vivo quantification of collagen in breast tissue

    NASA Astrophysics Data System (ADS)

    Taroni, Paola; Pifferi, Antonio; Quarto, Giovanna; Farina, Andrea; Ieva, Francesca; Paganoni, Anna Maria; Abbate, Francesca; Cassano, Enrico; Cubeddu, Rinaldo

    2015-05-01

    Time-resolved diffuse optical spectroscopy provides non-invasively the optical characterization of highly diffusive media, such as biological tissues. Light pulses are injected into the tissue and the effects of light propagation on re-emitted pulses are interpreted with the diffusion theory to assess simultaneously tissue absorption and reduced scattering coefficients. Performing spectral measurements, information on tissue composition and structure is derived applying the Beer law to the measured absorption and an empiric approximation to Mie theory to the reduced scattering. The absorption properties of collagen powder were preliminarily measured in the range of 600-1100 nm using a laboratory set-up for broadband time-resolved diffuse optical spectroscopy. Optical projection images were subsequently acquired in compressed breast geometry on 218 subjects, either healthy or bearing breast lesions, using a portable instrument for optical mammography that operates at 7 wavelengths selected in the range 635-1060 nm. For all subjects, tissue composition was estimated in terms of oxy- and deoxy-hemoglobin, water, lipids, and collagen. Information on tissue microscopic structure was also derived. Good correlation was obtained between mammographic breast density (a strong risk factor for breast cancer) and an optical index based on collagen content and scattering power (that accounts mostly for tissue collagen). Logistic regression applied to all optically derived parameters showed that subjects at high risk for developing breast cancer for their high breast density can effectively be identified based on collagen content and scattering parameters. Tissue composition assessed in breast lesions with a perturbative approach indicated that collagen and hemoglobin content are significantly higher in malignant lesions than in benign ones.

  7. Quantification of Protein Signatures in Archived Human Prostate Tissues Using Shotgun Proteomic Methods

    DTIC Science & Technology

    2012-05-01

    Fig. 2). The detergent-solubilized proteins were subsequently incubated with wheat - germ agglutinin (WGA) and concanavalin-A (ConA) beads, washed...optimizing protocols to extract and profile proteins in matched normal and diseased tissue samples using directed mass spectrometry methods. The ultimate...able to extract up to 100 micrograms of total protein using whole- mount FFPE RP tissue block samples. This Figure 2. Silver-stained SDS-PAGE gel

  8. Digital separation of diaminobenzidine-stained tissues via an automatic color-filtering for immunohistochemical quantification.

    PubMed

    Fu, Rong; Ma, Xiaomian; Bian, Zhaoying; Ma, Jianhua

    2015-02-01

    The digital separation of diaminobenzidine (DAB)-stained tissues from hematoxylin background is an important pre-processing step to analyze immunostains. In most stain separation methods, specific color channels (for example: RGB, HSI, CMYK) or color deconvolution matrices are used to obtain different tissue contrasts between DAB- and hematoxylin-stained areas. However, these methods could produce incomplete separation or color changes because the color spectra of stains and co-localized stains overlap in histological images. Therefore, we proposed an automatic color-filtering to separate hematoxylin- and DAB-stained tissues. In implantation, the RGB images of DAB-labeled immunostains are first converted to 8-bit BN images by a mathematical translation to produce the largest contrast between brown DAB-stained tissues and blue hematoxylin-stained tissues. The first valley in the histogram revised by nonuniform quantization is set as the cut-off point to obtain a brown filter. DAB-stained tissues are accurately delineated from the background counterstain, resulting in DAB-only-image and De-DAB-image. Subsequently, a blue filter is designed in the CIE-Lab color space to further delineate the hematoxylin-stained tissues from the De-DAB-image. Finally, the average values of the remaining pixels of the De-DAB-image are set as the background color of the DAB-only-image to manage uneven dyeing and provide DAB-stained-image for adaptive immunohistochemistry quantitation. Extensive experimental results demonstrated that the proposed method has significant advantages compared with existing methods in terms of complete stain separation without changing the color in DAB-stained areas.

  9. Quantification of phenylethylamine and p-tyramine in rat tissues using a new radioenzymatic assay

    SciTech Connect

    Hamburger, S.A.; Henry, D.P.

    1986-03-05

    Phenylethylamine (PEA) and p-tyramine (p-tym) are biologically active aralkylamines that are found in a number of mammalian tissues, including brain and plasma. The investigation of the biological role of these substances has been hampered by the lack of accessible assay methodology. They have developed a new radioenzymatic assay using barley root tyramine N-methyltransferase and tritiated S-adenosylmethionine. The products formed by the reaction are isolated by TLC. The assay sensitivity was 2.1 and 1.0 pg/tube for PEA and p-tym, respectively. The concentration of PEA and p-tym was determined simultaneously in tissues from Sprague-Dawley rats (280 gm). Plasma PEA and p-tym were 478 +/- 66 and 309 +/- 69 pg/ml, respectively. They conclude that this new procedure is applicable to all tissues examined in that all tissues contain both PEA and p-tym and that these amines are heterogeneously distributed in rat tissues.

  10. Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells.

    PubMed

    Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

    2016-04-11

    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.

  11. 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

  12. 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.

  13. Bone Tissue Properties Measurement by Reference Point Indentation in Glucocorticoid-Induced Osteoporosis.

    PubMed

    Mellibovsky, Leonardo; Prieto-Alhambra, Daniel; Mellibovsky, Fernando; Güerri-Fernández, Roberto; Nogués, Xavier; Randall, Connor; Hansma, Paul K; Díez-Perez, Adolfo

    2015-09-01

    Glucocorticoids, widely used in inflammatory disorders, rapidly increase bone fragility and, therefore, fracture risk. However, common bone densitometry measurements are not sensitive enough to detect these changes. Moreover, densitometry only partially recognizes treatment-induced fracture reductions in osteoporosis. Here, we tested whether the reference point indentation technique could detect bone tissue property changes early after glucocorticoid treatment initiation. After initial laboratory and bone density measurements, patients were allocated into groups receiving calcium + vitamin D (Ca+D) supplements or anti-osteoporotic drugs (risedronate, denosumab, teriparatide). Reference point indentation was performed on the cortical bone layer of the tibia by a handheld device measuring bone material strength index (BMSi). Bone mineral density was measured by dual-energy X-ray absorptiometry (DXA). Although Ca+D-treated patients exhibited substantial and significant deterioration, risedronate-treated patients exhibited no significant change, and both denosumab- and teriparatide-treated participants exhibited significantly improved BMSi 7 weeks after initial treatment compared with baseline; these trends remained stable for 20 weeks. In contrast, no densitometry changes were observed during this study period. In conclusion, our study is the first to our knowledge to demonstrate that reference point indentation is sensitive enough to reflect changes in cortical bone indentation after treatment with osteoporosis therapies in patients newly exposed to glucocorticoids.

  14. An HPLC-ECD method for monoamines and metabolites quantification in cuttlefish (cephalopod) brain tissue.

    PubMed

    Bidel, Flavie; Corvaisier, Sophie; Jozet-Alves, Christelle; Pottier, Ivannah; Dauphin, François; Naud, Nadège; Bellanger, Cécile

    2016-08-01

    The cuttlefish belongs to the mollusk class Cephalopoda, considered as the most advanced marine invertebrates and thus widely used as models to study the biology of complex behaviors and cognition, as well as their related neurochemical mechanisms. Surprisingly, methods to quantify the biogenic monoamines and their metabolites in cuttlefish brain remain sparse and measure a limited number of analytes. This work aims to validate an HPLC-ECD method for the simultaneous quantification of dopamine, serotonin, norepinephrine and their main metabolites in cuttlefish brain. In comparison and in order to develop a method suitable to answer both ecological and biomedical questions, the validation was also carried out on a phylogenetically remote species: mouse (mammals). The method was shown to be accurate, precise, selective, repeatable and sensitive over a wide range of concentrations for 5-hydroxyindole-3-acetic acid, serotonin, dopamine, 3,4-dihydroxyphenylacetic acid and norepinephrine in the both extracts of cuttlefish and mouse brain, though with low precision and recovery for 4-hydroxy-3-methoxyphenylethylene glycol. Homovanillic acid, accurately studied in rodents, was not detectable in the brain of cuttlefish. Overall, we described here the first fully validated HPLC method for the routine measurement of both monoamines and metabolites in cuttlefish brain. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Quantification of Interfibrillar Shear Stress in Aligned Soft Collagenous Tissues via Notch Tension Testing

    PubMed Central

    Szczesny, Spencer E.; Caplan, Jeffrey L.; Pedersen, Pal; Elliott, Dawn M.

    2015-01-01

    The mechanical function of soft collagenous tissues is largely determined by their hierarchical organization of collagen molecules. While collagen fibrils are believed to be discontinuous and transfer load through shearing of the interfibrillar matrix, interfibrillar shear stresses have never been quantified. Scaling traditional shear testing procedures down to the fibrillar length scale is impractical and would introduce substantial artifacts. Here, through the use of a novel microscopic variation of notch tension testing, we explicitly demonstrate the existence of interfibrillar shear stresses within tendon fascicles and provide the first measurement of their magnitude. Axial stress gradients along the sample length generated by notch tension testing were measured and used to calculate a value of 32 kPa for the interfibrillar shear stress. This estimate is comparable to the interfibrillar shear stress predicted by previous multiscale modeling of tendon fascicles, which supports the hypothesis that fibrils are discontinuous and transmit load through interfibrillar shear. This information regarding the structure-function relationships of tendon and other soft collagenous tissues is necessary to identify potential causes for tissue impairment with degeneration and provide the foundation for developing regenerative repair strategies or engineering biomaterials for tissue replacement. PMID:26469396

  16. Quantification of Interfibrillar Shear Stress in Aligned Soft Collagenous Tissues via Notch Tension Testing

    NASA Astrophysics Data System (ADS)

    Szczesny, Spencer E.; Caplan, Jeffrey L.; Pedersen, Pal; Elliott, Dawn M.

    2015-10-01

    The mechanical function of soft collagenous tissues is largely determined by their hierarchical organization of collagen molecules. While collagen fibrils are believed to be discontinuous and transfer load through shearing of the interfibrillar matrix, interfibrillar shear stresses have never been quantified. Scaling traditional shear testing procedures down to the fibrillar length scale is impractical and would introduce substantial artifacts. Here, through the use of a novel microscopic variation of notch tension testing, we explicitly demonstrate the existence of interfibrillar shear stresses within tendon fascicles and provide the first measurement of their magnitude. Axial stress gradients along the sample length generated by notch tension testing were measured and used to calculate a value of 32 kPa for the interfibrillar shear stress. This estimate is comparable to the interfibrillar shear stress predicted by previous multiscale modeling of tendon fascicles, which supports the hypothesis that fibrils are discontinuous and transmit load through interfibrillar shear. This information regarding the structure-function relationships of tendon and other soft collagenous tissues is necessary to identify potential causes for tissue impairment with degeneration and provide the foundation for developing regenerative repair strategies or engineering biomaterials for tissue replacement.

  17. Optical spectroscopy for quantification of bulk breast tissue properties in adolescent girls: preliminary observations

    NASA Astrophysics Data System (ADS)

    Dick, Samantha N.; Lilge, Lothar

    2005-09-01

    Optical technology holds considerable promise to improve early detection, diagnosis and risk assessment of breast cancer. Unlike current clinical risk assessment tools such as the Gail model, the most widely accepted risk assessment tool, optical risk assessment technology can be applied to the entire female population of all ages. This study is investigating the use of optical reflectance spectroscopy (ORS) as a possible breast tissue development monitoring tool for adolescent girls. Changes in breast development due to proliferation of mammary gland and the surrounding stroma are reflected in changes in breast tissue density and composition which can be interrogated optically. Modifications of development influenced by micronutrients and hormonal status from exposures (e.g. toxins), lifestyle and diet effects, may ultimately be tracked. Preliminary data suggests that ORS has the ability to detect differences in bulk tissue properties in the developing breast of adolescent girls when compared to developmental stages assessed by Tanner, regional variation within breast tissue structure and asymmetries between left and right breast size and shape. Spectral comparison of unilateral breast development permits adjusting the optode separation as function of developmental breast size to minimize optical sampling of pectoral muscle.

  18. Detection and quantification of virulent Aeromonas hydrophila in channel catfish tissues following waterborne challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to understand the pathogenesis of motile aeromonas septicemia caused by virulent A. hydrophila (vAh) in channel catfish, Ictalurus punctatus. Adipose fin clipped catfish were challenged with vAh using waterborne challenge method and the distribution of vAh in catfish tissue...

  19. Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material.

    PubMed

    Deprez, Liesbet; Corbisier, Philippe; Kortekaas, Anne-Marie; Mazoua, Stéphane; Beaz Hidalgo, Roxana; Trapmann, Stefanie; Emons, Hendrik

    2016-09-01

    Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use. Here, the results of an in-house validation of a droplet digital PCR method are presented. This method is intended for the quantification of the absolute copy number concentration of a purified linearized plasmid in solution with a nucleic acid background. It has been investigated which factors within the measurement process have a significant effect on the measurement results, and the contribution to the overall measurement uncertainty has been estimated. A comprehensive overview is provided on all the aspects that should be investigated when performing an in-house method validation of a digital PCR method.

  20. Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard.

    PubMed

    Toyota, Akie; Akiyama, Hiroshi; Sugimura, Mitsunori; Watanabe, Takahiro; Kikuchi, Hiroyuki; Kanamori, Hisayuki; Hino, Akihiro; Esaka, Muneharu; Maitani, Tamio

    2006-04-01

    Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.

  1. Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection.

    PubMed

    Caldas, Sérgio; Caldas, Ivo Santana; Diniz, Lívia de Figueiredo; Lima, Wanderson Geraldo de; Oliveira, Riva de Paula; Cecílio, Alzira Batista; Ribeiro, Isabela; Talvani, André; Bahia, Maria Terezinha

    2012-09-01

    The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p<0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our

  2. Non-Invasive Quantification of White and Brown Adipose Tissues and Liver Fat Content by Computed Tomography in Mice

    PubMed Central

    Lubura, Marko; Hesse, Deike; Neumann, Nancy; Scherneck, Stephan; Wiedmer, Petra; Schürmann, Annette

    2012-01-01

    Objectives Obesity and its distribution pattern are important factors for the prediction of the onset of diabetes in humans. Since several mouse models are suitable to study the pathophysiology of type 2 diabetes the aim was to validate a novel computed tomograph model (Aloka-Hitachi LCT-200) for the quantification of visceral, subcutaneous, brown and intrahepatic fat depots in mice. Methods Different lean and obese mouse models (C57BL/6, B6.V-Lepob, NZO) were used to determine the most adequate scanning parameters for the detection of the different fat depots. The data were compared with those obtained after preparation and weighing the fat depots. Liver fat content was determined by biochemical analysis. Results The correlations between weights of fat tissues on scale and weights determined by CT were significant for subcutaneous (r2 = 0.995), visceral (r2 = 0.990) and total white adipose tissue (r2 = 0.992). Moreover, scans in the abdominal region, between lumbar vertebrae L4 to L5 correlated with whole-body fat distribution allowing experimenters to reduce scanning time and animal exposure to radiation and anesthesia. Test-retest reliability and measurements conducted by different experimenters showed a high reproducibility in the obtained results. Intrahepatic fat content estimated by CT was linearly related to biochemical analysis (r2 = 0.915). Furthermore, brown fat mass correlated well with weighted brown fat depots (r2 = 0.952). In addition, short-term cold-expose (4°C, 4 hours) led to alterations in brown adipose tissue attributed to a reduction in triglyceride content that can be visualized as an increase in Hounsfield units by CT imaging. Conclusion The 3D imaging of fat by CT provides reliable results in the quantification of total, visceral, subcutaneous, brown and intrahepatic fat in mice. This non-invasive method allows the conduction of longitudinal studies of obesity in mice and therefore enables experimenters to investigate

  3. Quantification of virus-like particles suggests viral infection in corals affected by Porites tissue loss

    NASA Astrophysics Data System (ADS)

    Lawrence, Scott A.; Davy, Joanne E.; Aeby, Greta S.; Wilson, William H.; Davy, Simon K.

    2014-09-01

    Porites tissue loss is a common disease of Porites compressa on Hawaiian reefs. Despite its prevalence, to date, the aetiological agent of the disease has not been found. The apparent lack of a microbial causative agent in the similar disease Porites bleaching with tissue loss, as well as increasing evidence of viral infections in scleractinian corals and Symbiodinium, led us to hypothesise that a virus may be responsible. Electron microscopy revealed the presence of numerous and varied virus-like particles (VLPs) in healthy and diseased P. compressa colonies. While overall virus numbers were similar in all samples, the abundance of a group of icosahedral VLPs differed significantly between healthy and diseased colonies. While not conclusive, these results suggest that viruses may play a role in this disease, and provide a basis for further studies.

  4. Quantification of phase retardation in corneal tissues using a femtosecond laser

    NASA Astrophysics Data System (ADS)

    Calhoun, William R.; Beylin, Alexander; Weiblinger, Richard; Ilev, Ilko

    2013-03-01

    The use of femtosecond lasers (FSL) in ophthalmic procedures, such as LASIK, lens replacement (cataract surgery), as well as several other treatments, is growing rapidly. The treatment effect is based on photo ablation of ocular tissues by a series of ultra-short laser pulses. However, the laser beam characteristics change dynamically due to interactions with birefringent corneal tissue, which may affect the outcome of the laser treatment. To better understand the effect the cornea has on the laser characteristics, we developed a system for measuring retardation and validated it with precise, standard phase retarders. Then we measured the phase retardation of FSLs through bovine corneas and found that there is a considerable, location dependent, variation in retardation values. This information can potentially help optimize FSL parameters to make their application in ophthalmic procedures safer and more effective.

  5. Enhancing the quantification of tissue sodium content by MRI: time-efficient sodium B1 mapping at clinical field strengths.

    PubMed

    Lommen, Jonathan; Konstandin, Simon; Krämer, Philipp; Schad, Lothar R

    2016-02-01

    Tissue sodium content (TSC) is a sensitive measure of pathological changes and can be detected non-invasively by MRI. For the absolute quantification of TSC, B1 inhomogeneities must be corrected, which is not well established beyond research applications. An in-depth analysis of B1 mapping methods which are suitable for application in TSC quantification is presented. On the basis of these results, a method for simultaneous B1 mapping and imaging is proposed in order to enhance accuracy and to reduce measurement time at clinical field strengths. The B1 mapping techniques used were phase-sensitive (PS), Bloch-Siegert shift (BSS), double-angle (DAM) and actual flip-angle imaging (AFI) methods. Experimental and theoretical comparisons demonstrated that the PS technique yields the most accurate field profiles and exhibits the highest signal-to-noise ratio (SNR). Simultaneous B1 mapping and imaging was performed for the PS method, employing both degrees of freedom of the MR signal: the B1 field is encoded into signal phase and the amplitude provides the concentration information. In comparison with the more established DAM, a 13% higher SNR was obtained and field effects could be corrected more accurately without the need for additional measurement time. The protocol developed was applied to measure TSC in the healthy human head at an isotropic resolution of 4 mm. TSC was determined to be 35 ± 1 mM in white matter and 134 ± 3 mM in vitreous humor. By employing the proposed simultaneous characterization of the B1 field and acquisition of the spin density-weighted sodium signal, the accuracy of the non-invasive measurement of TSC is enhanced and the measurement time is reduced. This should allow (23)Na MRI to be better incorporated into clinical studies and routine.

  6. Quantification of Protein Signatures in Archived Human Prostate Tissues Using Shotgun Proteomic Methods

    DTIC Science & Technology

    2011-06-01

    proteins were subsequently incubated with wheat - germ agglutinin (WGA) and con canavalin- A (ConA) beads, washed, and el uted with sol uble n-acetyl...prostate radial prostatectomies and have focused on optimizing protocols to extract and profile proteins in matched normal and dise ased tissue s...human prostate cancer using label-free, quantitative mass spectrometry. Last year we were able to extract up to 1 00 micrograms of total protein

  7. Evaluation and quantification of spectral information in tissue by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Maeder, Ulf; Marquardt, Kay; Beer, Sebastian; Bergmann, Thorsten; Schmidts, Thomas; Heverhagen, Johannes T.; Zink, Klemens; Runkel, Frank; Fiebich, Martin

    2012-10-01

    A confocal imaging and image processing scheme is introduced to visualize and evaluate the spatial distribution of spectral information in tissue. The image data are recorded using a confocal laser-scanning microscope equipped with a detection unit that provides high spectral resolution. The processing scheme is based on spectral data, is less error-prone than intensity-based visualization and evaluation methods, and provides quantitative information on the composition of the sample. The method is tested and validated in the context of the development of dermal drug delivery systems, introducing a quantitative uptake indicator to compare the performances of different delivery systems is introduced. A drug penetration study was performed in vitro. The results show that the method is able to detect, visualize and measure spectral information in tissue. In the penetration study, uptake efficiencies of different experiment setups could be discriminated and quantitatively described. The developed uptake indicator is a step towards a quantitative assessment and, in a more general view apart from pharmaceutical research, provides valuable information on tissue composition. It can potentially be used for clinical in vitro and in vivo applications.

  8. New method of dynamic color doppler signal quantification in metastatic lymph nodes compared to direct polarographic measurements of tissue oxygenation.

    PubMed

    Scholbach, Thomas; Scholbach, Jakob; Krombach, Gabriele A; Gagel, Bernd; Maneschi, Payam; Di Martino, Ercole

    2005-05-10

    Tumor growth depends on sufficient blood and oxygen supply. Hypoxia stimulates neovascularization and is a known cause for radio- and chemoresistance. The objective of this study was to investigate the use of a novel ultrasound technique for the dynamic assessment of vascularization and oxygenation in metastatic lymph nodes. Twenty-four patients (age 44-78 years) with cervical lymph node metastases of squamous cell head and neck cancer were investigated by color duplex sonography and 17 (age 46-78 years) were investigated additionally with polarography. Sonography was performed after contrast enhancer infusion under defined conditions. Intranodal perfusion data (color hue, colored area) were measured automatically by a novel software technique. This allows an evaluation of blood flow dynamics by calculating perfusion intensity--velocity, perfused area, as well as the novel parameters tissue resistance index (TRI) and tissue pulsatility index (TPI)--for each point of a complete heart cycle. Tumor tissue pO(2) was measured by means of polarographic needle electrodes placed intranodally. The sonographic and polarographic data were correlated using Pearson's test. Sonography demonstrated a statistically significant inverse correlation between hypoxia and perfusion and significant TPI and TRI changes with different N-stages. The percentage of nodal fraction with less than 10 mmHg oxygen saturation was significantly inversely correlated with lymph node perfusion (r = -0.551; p = 0.021). Nodes with a perfusion of less than 0.05 cm/sec flow velocity showed significantly larger hypoxic areas (p = 0.006). Significant differences of TPI and TRI existed between nodes in stage N(1) and N(2)/N(3) (p = 0.028 and 0.048, respectively). This new method of dynamic signal quantification allows a noninvasive and quantitative assessment of tumor and metastatic lymph node perfusion by means of commonly available ultrasound equipment.

  9. Three-dimensional pore space quantification of apple tissue using X-ray computed microtomography.

    PubMed

    Mendoza, Fernando; Verboven, Pieter; Mebatsion, Hibru K; Kerckhofs, Greet; Wevers, Martine; Nicolaï, Bart

    2007-08-01

    The microstructure and the connectivity of the pore space are important variables for better understanding of the complex gas transport phenomena that occur in plant tissues. In this study, we present an experimental procedure for image acquisition and image processing to quantitatively characterize in 3D the pore space of apple tissues (Malus domestica Borkh.) for two cultivars (Jonagold and Braeburn) taken from the fleshy part of the cortex using X-ray computer microtomography. Preliminary sensitivity analyses were performed to determine the effect of the resolution and the volume size (REV, representative elementary volume analysis) on the computed porosity of apple samples. For comparison among cultivars, geometrical properties such as porosity, specific surface area, number of disconnected pore volumes and their distribution parameters were extracted and analyzed in triplicate based on the 3D skeletonization of the pore space (medial axis analysis). The results showed that microtomography provides a resolution at the micrometer level to quantitatively analyze and characterize the 3D topology of the pore space in apple tissue. The computed porosity was confirmed to be highly dependent of the resolution used, and the minimum REV of the cortical flesh of apple fruit was estimated to be 1.3 mm(3). Comparisons among the two cultivars using a resolution of 8.5 mum with a minimum REV cube showed that in spite of the complexity and variability of the pore space network observed in Jonagold and Braeburn apples, the extracted parameters from the medial axis were significantly different (P-value < 0.05). Medial axis parameters showed potential to differentiate the microstructure between the two evaluated apple cultivars.

  10. Extraction and quantification of adenosine triphosphate in mammalian tissues and cells.

    PubMed

    Chida, Junji; Kido, Hiroshi

    2014-01-01

    Adenosine 5'-triphosphate (ATP) is the "energy currency" of organisms and plays central roles in bioenergetics, whereby its level is used to evaluate cell viability, proliferation, death, and energy transmission. In this chapter, we describe an improved and efficient method for extraction of ATP from tissues and cells using phenol-based reagents. The chaotropic extraction reagents reported so far co-precipitate ATP with insoluble proteins during extraction and with salts during neutralization. In comparison, the phenol-based reagents extract ATP well without the risks of co-precipitation. The extracted ATP can be quantified by the luciferase assay or high-performance liquid chromatography.

  11. Quantification of major flavonoids in carnation tissues (Dianthus caryophyllus) as a tool for cultivar discrimination.

    PubMed

    Galeotti, Francesco; Barile, Elisa; Lanzotti, Virginia; Dolci, Marcello; Curir, Paolo

    2008-01-01

    One flavone-C-glycoside and two flavonol-O-glycosides were recognized and isolated as the main flavonoidal components in nine different carnation cultivars, and their chemical structures have been determined by spectroscopic methods, including UV detection, MS and NMR. The distribution of these three compounds in flowers, leaves, stems, young sprouts, and roots of each cultivar was evaluated by a simple HPLC-UV method: the graphic representation of their content in the different tissues allows to identify and characterize unambiguously each considered carnation cultivar. The presented method could be an easy, inexpensive and reliable tool for carnation cultivar discrimination.

  12. Measurement and quantification of fluorescent changes in ocular tissue using a novel confocal instrument

    NASA Astrophysics Data System (ADS)

    Buttenschoen, Kim K.; Girkin, John M.; Daly, Daniel J.

    2014-05-01

    Our sight is a major contributor to our quality of life. The treatment of diseases like macular degeneration and glaucoma, however, presents a challenge as the delivery of medication to ocular tissue is not well understood. The instrument described here will help quantify targeted delivery by non-invasively and simultaneously measuring light reflected from and fluorescence excited in the eye, used as position marker and to track compounds respectively. The measurement concept has been proven by monitoring the diffusion of fluorescein and a pharmaceutical compound for treating open angle glaucoma in vitro in a cuvette and in ex vivo porcine eyes. To obtain a baseline of natural fluorescence we measured the change in corneal and crystalline lens autofluorescence in volunteers over a week. We furthermore present data on 3D ocular autofluorescence. Our results demonstrate the capability to measure the location and concentration of the compound of interest with high axial and temporal resolution of 178 μm and 0.6 s respectively. The current detection limit is 2 nM for fluorescein, and compounds with a quantum yield as low as 0.01 were measured to concentrations below 1 μM. The instrument has many applications in assessing the diffusion of fluorescent compounds through the eye and skin in vitro and in vivo, measuring autofluorescence of ocular tissues and reducing the number of animals needed for research. The instrument has the capability of being used both in the clinical and home care environment opening up the possibility of measuring controlled drug release in a patient friendly manner.

  13. Insights into reference point indentation involving human cortical bone: sensitivity to tissue anisotropy and mechanical behavior.

    PubMed

    Granke, Mathilde; Coulmier, Aurélie; Uppuganti, Sasidhar; Gaddy, Jennifer A; Does, Mark D; Nyman, Jeffry S

    2014-09-01

    Reference point indentation (RPI) is a microindentation technique involving 20 cycles of loading in "force-control" that can directly assess a patient׳s bone tissue properties. Even though preliminary clinical studies indicate a capability for fracture discrimination, little is known about what mechanical behavior the various RPI properties characterize and how these properties relate to traditional mechanical properties of bone. To address this, the present study investigated the sensitivity of RPI properties to anatomical location and tissue organization as well as examined to what extent RPI measurements explain the intrinsic mechanical properties of human cortical bone. Multiple indents with a target force of 10N were done in 2 orthogonal directions (longitudinal and transverse) per quadrant (anterior, medial, posterior, and lateral) of the femoral mid-shaft acquired from 26 donors (25-101 years old). Additional RPI measurements were acquired for 3 orthogonal directions (medial only). Independent of age, most RPI properties did not vary among these locations, but they did exhibit transverse isotropy such that resistance to indentation is greater in the longitudinal (axial) direction than in the transverse direction (radial or circumferential). Next, beam specimens (~2mm×5mm×40mm) were extracted from the medial cortex of femoral mid-shafts, acquired from 34 donors (21-99 years old). After monotonically loading the specimens in three-point bending to failure, RPI properties were acquired from an adjacent region outside the span. Indent direction was orthogonal to the bending axis. A significant inverse relationship was found between resistance to indentation and the apparent-level mechanical properties. Indentation distance increase (IDI) and a linear combination of IDI and the loading slope, averaged over cycles 3 through 20, provided the best explanation of the variance in ultimate stress (r(2)=0.25, p=0.003) and toughness (r(2)=0.35, p=0.004), respectively

  14. Implementation of an absolute brain 1H-MRS quantification method to assess different tissue alterations in multiple sclerosis.

    PubMed

    Bagory, Matthieu; Durand-Dubief, Françoise; Ibarrola, Danielle; Comte, Jean-Christophe; Cotton, François; Confavreux, Christian; Sappey-Marinier, Dominique

    2012-10-01

    Magnetic resonance spectroscopy has emerged as a sensitive modality to detect early and diffuse alterations in multiple sclerosis. Recently, the hypothesis of neurodegenerative pathogenesis has highlightened the interest for measurement of metabolites concentrations, to gain specificity, in a large brain volume encompassing different tissue alterations. Therefore, we proposed in this paper the implementation of an absolute quantification method based on localized spectroscopy at short (30 ms) and long (135 ms) echo time of a volume including normal appearing white matter, cortical gray matter, and lesions. First, methodological developments were implemented including external calibration, and corrections of phased-array coil sensitivity and cerebrospinal fluid volume contribution. Second, these improvements were validated and optimized using an original methodology based on simulations of brain images with lesions. Finally, metabolic alterations were assessed in 65 patients including 26 relapsing-remitting, 17 primary-progressive (PP), 22 secondary-progressive (SP) patients, and in 23 normal subjects. Results showed increases of choline, creatine, and myo-inositol concentrations in PP and SP patients compared to controls, whereas the concentration of N-acetyl compounds remained constant. The major finding of this study was the identification of Cho concentration and Cho/tNA ratio as putative markers of progressive onset, suggesting interesting perspectives in detection and followup of neurodegenerative processes.

  15. Model-based cell number quantification using online single-oxygen sensor data for tissue engineering perfusion bioreactors.

    PubMed

    Lambrechts, T; Papantoniou, I; Sonnaert, M; Schrooten, J; Aerts, J-M

    2014-10-01

    Online and non-invasive quantification of critical tissue engineering (TE) construct quality attributes in TE bioreactors is indispensable for the cost-effective up-scaling and automation of cellular construct manufacturing. However, appropriate monitoring techniques for cellular constructs in bioreactors are still lacking. This study presents a generic and robust approach to determine cell number and metabolic activity of cell-based TE constructs in perfusion bioreactors based on single oxygen sensor data in dynamic perfusion conditions. A data-based mechanistic modeling technique was used that is able to correlate the number of cells within the scaffold (R(2)  = 0.80) and the metabolic activity of the cells (R(2)  = 0.82) to the dynamics of the oxygen response to step changes in the perfusion rate. This generic non-destructive measurement technique is effective for a large range of cells, from as low as 1.0 × 10(5) cells to potentially multiple millions of cells, and can open-up new possibilities for effective bioprocess monitoring.

  16. Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

    PubMed Central

    Song, Alice Tung Wan; de Mello, Evandro Sobroza; Alves, Venâncio Avancini Ferreira; Cavalheiro, Norma de Paula; Melo, Carlos Eduardo; Bonazzi, Patricia Rodrigues; Tengan, Fatima Mitiko; Freire, Maristela Pinheiro; Barone, Antonio Alci; D'Albuquerque, Luiz Augusto Carneiro; Abdala, Edson

    2015-01-01

    Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection. PMID:25742264

  17. Optical Quantification of Harmonic Acoustic Radiation Force Excitation in a Tissue-Mimicking Phantom.

    PubMed

    Suomi, Visa; Edwards, David; Cleveland, Robin

    2015-12-01

    Optical tracking was used to characterize acoustic radiation force-induced displacements in a tissue-mimicking phantom. Amplitude-modulated 3.3-MHz ultrasound was used to induce acoustic radiation force in the phantom, which was embedded with 10-μm microspheres that were tracked using a microscope objective and high-speed camera. For sine and square amplitude modulation, the harmonic components of the fundamental and second and third harmonic frequencies were measured. The displacement amplitudes were found to increase linearly with acoustic radiation force up to 10 μm, with sine modulation having 19.5% lower peak-to-peak amplitude values than square modulation. Square modulation produced almost no second harmonic, but energy was present in the third harmonic. For the sine modulation, energy was present in the second harmonic and low energy in the third harmonic. A finite-element model was used to simulate the deformation and was both qualitatively and quantitatively in agreement with the measurements.

  18. Identification of stable reference genes for gene expression analysis of three-dimensional cultivated human bone marrow-derived mesenchymal stromal cells for bone tissue engineering.

    PubMed

    Rauh, Juliane; Jacobi, Angela; Stiehler, Maik

    2015-02-01

    The principles of tissue engineering (TE) are widely used for bone regeneration concepts. Three-dimensional (3D) cultivation of autologous human mesenchymal stromal cells (MSCs) on porous scaffolds is the basic prerequisite to generate newly formed bone tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive analytical tool for the measurement of mRNA-levels in cells or tissues. For an accurate quantification of gene expression levels, stably expressed reference genes (RGs) are essential to obtain reliable results. Since the 3D environment can affect a cell's morphology, proliferation, and gene expression profile compared with two-dimensional (2D) cultivation, there is a need to identify robust RGs for the quantification of gene expression. So far, this issue has not been adequately investigated. The aim of this study was to identify the most stably expressed RGs for gene expression analysis of 3D-cultivated human bone marrow-derived MSCs (BM-MSCs). For this, we analyzed the gene expression levels of n=31 RGs in 3D-cultivated human BM-MSCs from six different donors compared with conventional 2D cultivation using qRT-PCR. MSCs isolated from bone marrow aspirates were cultivated on human cancellous bone cube scaffolds for 14 days. Osteogenic differentiation was assessed by cell-specific alkaline phosphatase (ALP) activity and expression of osteogenic marker genes. Expression levels of potential reference and target genes were quantified using commercially available TaqMan(®) assays. mRNA expression stability of RGs was determined by calculating the coefficient of variation (CV) and using the algorithms of geNorm and NormFinder. Using both algorithms, we identified TATA box binding protein (TBP), transferrin receptor (p90, CD71) (TFRC), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the most stably expressed RGs in 3D-cultivated BM-MSCs. Notably, genes that are routinely used as RGs, for example, beta actin

  19. Selection of suitable reference genes for gene expression studies in normal human ovarian tissues, borderline ovarian tumours and ovarian cancer.

    PubMed

    Ofinran, Olumide; Bose, Ujjal; Hay, Daniel; Abdul, Summi; Tufatelli, Cristina; Khan, Raheela

    2016-12-01

    The use of reference genes is the most common method of controlling the variation in mRNA expression during quantitative polymerase chain reaction, although the use of traditional reference genes, such as β‑actin, glyceraldehyde‑3‑phosphate dehydrogenase or 18S ribosomal RNA, without validation occasionally leads to unreliable results. Therefore, the present study aimed to evaluate a set of five commonly used reference genes to determine the most suitable for gene expression studies in normal ovarian tissues, borderline ovarian and ovarian cancer tissues. The expression stabilities of these genes were ranked using two gene stability algorithms, geNorm and NormFinder. Using geNorm, the two best reference genes in ovarian cancer were β‑glucuronidase and β‑actin. Hypoxanthine phosphoribosyltransferase‑1 and β‑glucuronidase were the most stable in ovarian borderline tumours, and hypoxanthine phosphoribosyltransferase‑1 and glyceraldehyde‑3‑phosphate dehydrogenase were the most stable in normal ovarian tissues. NormFinder ranked β‑actin the most stable in ovarian cancer, and the best combination of two genes was β‑glucuronidase and β‑actin. In borderline tumours, hypoxanthine phosphoribosyltransferase‑1 was identified as the most stable, and the best combination was hypoxanthine phosphoribosyltransferase‑1 and β‑glucuronidase. In normal ovarian tissues, β‑glucuronidase was recommended as the optimum reference gene, and the most optimum pair of reference genes was hypoxanthine phosphoribosyltransferase‑1 and β‑actin. To the best of our knowledge, this is the first study to investigate the selection of a set of reference genes for normalisation in quantitative polymerase chain reactions in different ovarian tissues, and therefore it is recommended that β‑glucuronidase, β‑actin and hypoxanthine phosphoribosyltransferase‑1 are the most suitable reference genes for such analyses.

  20. Freeze-drying for the stabilisation of shellfish toxins in mussel tissue (Mytilus edulis) reference materials.

    PubMed

    McCarron, Pearse; Emteborg, Håkan; Hess, Philipp

    2007-04-01

    Aliquots of freeze-dried and wet mussel tissue reference materials containing the various shellfish toxins examined in the study.

  1. A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue.

    PubMed

    Azaripour, Adriano; Lagerweij, Tonny; Scharfbillig, Christina; Jadczak, Anna Elisabeth; Willershausen, Brita; Van Noorden, Cornelis J F

    2016-08-01

    For 3-dimensional (3D) imaging of a tissue, 3 methodological steps are essential and their successful application depends on specific characteristics of the type of tissue. The steps are 1° clearing of the opaque tissue to render it transparent for microscopy, 2° fluorescence labeling of the tissues and 3° 3D imaging. In the past decades, new methodologies were introduced for the clearing steps with their specific advantages and disadvantages. Most clearing techniques have been applied to the central nervous system and other organs that contain relatively low amounts of connective tissue including extracellular matrix. However, tissues that contain large amounts of extracellular matrix such as dermis in skin or gingiva are difficult to clear. The present survey lists methodologies that are available for clearing of tissues for 3D imaging. We report here that the BABB method using a mixture of benzyl alcohol and benzyl benzoate and iDISCO using dibenzylether (DBE) are the most successful methods for clearing connective tissue-rich gingiva and dermis of skin for 3D histochemistry and imaging of fluorescence using light-sheet microscopy.

  2. Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

    PubMed

    Yang, Zhimin; Chen, Yu; Hu, Baoyun; Tan, Zhiqun; Huang, Bingru

    2015-01-01

    Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

  3. Identification and Validation of Reference Genes for Quantification of Target Gene Expression with Quantitative Real-time PCR for Tall Fescue under Four Abiotic Stresses

    PubMed Central

    Hu, Baoyun; Tan, Zhiqun; Huang, Bingru

    2015-01-01

    Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species. PMID:25786207

  4. AtRTD - a comprehensive reference transcript dataset resource for accurate quantification of transcript-specific expression in Arabidopsis thaliana.

    PubMed

    Zhang, Runxuan; Calixto, Cristiane P G; Tzioutziou, Nikoleta A; James, Allan B; Simpson, Craig G; Guo, Wenbin; Marquez, Yamile; Kalyna, Maria; Patro, Rob; Eyras, Eduardo; Barta, Andrea; Nimmo, Hugh G; Brown, John W S

    2015-10-01

    RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.

  5. Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale

    PubMed Central

    Cross, N C P; White, H E; Ernst, T; Welden, L; Dietz, C; Saglio, G; Mahon, F-X; Wong, C C; Zheng, D; Wong, S; Wang, S-S; Akiki, S; Albano, F; Andrikovics, H; Anwar, J; Balatzenko, G; Bendit, I; Beveridge, J; Boeckx, N; Cerveira, N; Cheng, S-M; Colomer, D; Czurda, S; Daraio, F; Dulucq, S; Eggen, L; El Housni, H; Gerrard, G; Gniot, M; Izzo, B; Jacquin, D; Janssen, J J W M; Jeromin, S; Jurcek, T; Kim, D-W; Machova-Polakova, K; Martinez-Lopez, J; McBean, M; Mesanovic, S; Mitterbauer-Hohendanner, G; Mobtaker, H; Mozziconacci, M-J; Pajič, T; Pallisgaard, N; Panagiotidis, P; Press, R D; Qin, Y-Z; Radich, J; Sacha, T; Touloumenidou, T; Waits, P; Wilkinson, E; Zadro, R; Müller, M C; Hochhaus, A; Branford, S

    2016-01-01

    Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR1–MR4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR4.5 level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms. PMID:27109508

  6. Quantification of the Iodine Content of Perigastric Adipose Tissue by Dual-Energy CT: A Novel Method for Preoperative Diagnosis of T4-Stage Gastric Cancer

    PubMed Central

    Shi, Gaofeng; Li, Yang; Li, Yong

    2015-01-01

    This study investigated the utility of quantifying iodine concentration (IC) in perigastric adipose tissue, using dual-energy computed tomography (DECT), for the detection of T4a-stage gastric cancer. Fifty-four patients with gastric cancer were enrolled at the Fourth Hospital of Hebei Medical University between January and June 2013. Patients were imaged preoperatively with conventional computed tomography (CT) scans and DECT, and the IC in perigastric fat adjacent to the tumor calculated from arterial phase (AP) and portal venous phase (PVP) images. The patients subsequently received surgical treatment (gastrectomy), and histologic analysis of resected specimens was used as a ‘gold standard’ reference for cancer staging. Receiver operating characteristic (ROC) curve analysis was employed to assess the utility of DECT for identifying T4a-stage gastric cancer, with optimal IC thresholds determined from the area under the ROC curve (AUC). Postoperative histology revealed that 32 patients had serosal invasion (group A), and 22 did not (group B). The accuracy of conventional CT for distinguishing stage T4 from non-T4 stages was 68.5% (37/54). IC was significantly higher in group A than in group B (AP: 0.60±0.34 vs. 0.09±0.19 mg/mL, p<0.001; PVP: 0.83±0.41 vs. 0.27±0.21 mg/mL, p<0.001). The sensitivity, specificity and AUC for detecting serosal invasion were 77.1%, 79.2% and 0.89 at an IC threshold of 0.25 mg/mL for AP images; and 80.0%, 79.2% and 0.90 at an IC threshold of 0.45 mg/mL for PVP images. These results indicated that Iodine quantification in perigastric fat using DECT is an accurate method for detecting serosal invasion by gastric cancer. PMID:26372042

  7. Poly(ADP-ribose): Structure, Physicochemical Properties and Quantification In Vivo, with Special Reference to Poly(ADP-ribose) Binding Protein Modules.

    PubMed

    Miwa, Masanao; Ida, Chieri; Yamashita, Sachiko; Tanaka, Masakazu; Fujisawa, Junichi

    2016-01-01

    PolyADP-ribosylation is a unique posttranslational modification of proteins, involved in various cellular functions including stability of chromatin. PolyADP-ribosylation modifies acceptor proteins with a large negatively charged poly(ADP-ribose) (PAR) to greatly change the structure and function of the acceptor proteins. In addition various specific motifs of proteins were recently found to interact non-covalently with PAR thereby changing the spaciotemporal activity of protein-protein interaction in cells. However, the structure of PAR to which specific protein motifs should bind is not fully characterized. The present work will review the structure, physicochemical properties and quantification of PAR in vivo, with special reference to PAR binding protein modules.

  8. DAKOTA, a multilevel parallel object-oriented framework for design optimization, parameter estimation, uncertainty quantification, and sensitivity analysis:version 4.0 reference manual

    SciTech Connect

    Griffin, Joshua D. (Sandai National Labs, Livermore, CA); Eldred, Michael Scott; Martinez-Canales, Monica L.; Watson, Jean-Paul; Kolda, Tamara Gibson; Adams, Brian M.; Swiler, Laura Painton; Williams, Pamela J.; Hough, Patricia Diane; Gay, David M.; Dunlavy, Daniel M.; Eddy, John P.; Hart, William Eugene; Guinta, Anthony A.; Brown, Shannon L.

    2006-10-01

    The DAKOTA (Design Analysis Kit for Optimization and Terascale Applications) toolkit provides a flexible and extensible interface between simulation codes and iterative analysis methods. DAKOTA contains algorithms for optimization with gradient and nongradient-based methods; uncertainty quantification with sampling, reliability, and stochastic finite element methods; parameter estimation with nonlinear least squares methods; and sensitivity/variance analysis with design of experiments and parameter study methods. These capabilities may be used on their own or as components within advanced strategies such as surrogate-based optimization, mixed integer nonlinear programming, or optimization under uncertainty. By employing object-oriented design to implement abstractions of the key components required for iterative systems analyses, the DAKOTA toolkit provides a flexible and extensible problem-solving environment for design and performance analysis of computational models on high performance computers. This report serves as a reference manual for the commands specification for the DAKOTA software, providing input overviews, option descriptions, and example specifications.

  9. DAKOTA : a multilevel parallel object-oriented framework for design optimization, parameter estimation, uncertainty quantification, and sensitivity analysis. Version 5.0, user's reference manual.

    SciTech Connect

    Eldred, Michael Scott; Dalbey, Keith R.; Bohnhoff, William J.; Adams, Brian M.; Swiler, Laura Painton; Hough, Patricia Diane; Gay, David M.; Eddy, John P.; Haskell, Karen H.

    2010-05-01

    The DAKOTA (Design Analysis Kit for Optimization and Terascale Applications) toolkit provides a flexible and extensible interface between simulation codes and iterative analysis methods. DAKOTA contains algorithms for optimization with gradient and nongradient-based methods; uncertainty quantification with sampling, reliability, and stochastic finite element methods; parameter estimation with nonlinear least squares methods; and sensitivity/variance analysis with design of experiments and parameter study methods. These capabilities may be used on their own or as components within advanced strategies such as surrogate-based optimization, mixed integer nonlinear programming, or optimization under uncertainty. By employing object-oriented design to implement abstractions of the key components required for iterative systems analyses, the DAKOTA toolkit provides a flexible and extensible problem-solving environment for design and performance analysis of computational models on high performance computers. This report serves as a reference manual for the commands specification for the DAKOTA software, providing input overviews, option descriptions, and example specifications.

  10. Quantification of the predominant monomeric catechins in baking chocolate standard reference material by LC/APCI-MS.

    PubMed

    Nelson, Bryant C; Sharpless, Katherine E

    2003-01-29

    Catechins are polyphenolic plant compounds (flavonoids) that may offer significant health benefits to humans. These benefits stem largely from their anticarcinogenic, antioxidant, and antimutagenic properties. Recent epidemiological studies suggest that the consumption of flavonoid-containing foods is associated with reduced risk of cardiovascular disease. Chocolate is a natural cocoa bean-based product that reportedly contains high levels of monomeric, oligomeric, and polymeric catechins. We have applied solid-liquid extraction and liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry to the identification and determination of the predominant monomeric catechins, (+)-catechin and (-)-epicatechin, in a baking chocolate Standard Reference Material (NIST Standard Reference Material 2384). (+)-Catechin and (-)-epicatechin are detected and quantified in chocolate extracts on the basis of selected-ion monitoring of their protonated [M + H](+) molecular ions. Tryptophan methyl ester is used as an internal standard. The developed method has the capacity to accurately quantify as little as 0.1 microg/mL (0.01 mg of catechin/g of chocolate) of either catechin in chocolate extracts, and the method has additionally been used to certify (+)-catechin and (-)-epicatechin levels in the baking chocolate Standard Reference Material. This is the first reported use of liquid chromatography/mass spectrometry for the quantitative determination of monomeric catechins in chocolate and the only report certifying monomeric catechin levels in a food-based Standard Reference Material.

  11. A candidate reference method for quantification of low concentrations of plasmid DNA by exhaustive counting of single DNA molecules in a flow stream

    NASA Astrophysics Data System (ADS)

    Yoo, Hee-Bong; Oh, Donggeun; Song, Jae Yong; Kawaharasaki, Mamoru; Hwang, Jeeseong; Yang, In Chul; Park, Sang-Ryoul

    2014-10-01

    This work demonstrates accurate measurement of the amount of substance concentration of low concentration plasmid DNA by counting individual DNA molecules using a high-sensitivity flow cytometric setup. Plasmid DNA is a widely used form of DNA, and its quantity often needs to be accurately determined. This work establishes a reference analytical method for direct quantification of low concentration plasmid DNA prepared as reference standards for polymerase chain reaction-based DNA quantification. The model plasmid DNA pBR322 (4361 bp) was stained with a fluorescent dye and was detected in a flow stream in a micro-fluidic channel with laser-induced fluorescence detection, for which the DNA flow was electro-hydrodynamically focused at the centre of the channel. 200 to 8000 DNA molecules in a ˜1 µL sample volume were counted within 2 min in an ‘exhaustive counting’ manner, which facilitated quantitation without calibration. The sample volume was measured and validated from the close agreement of the results of two independent measurement methods, gravimetric determination of water filling the capillary and graphical estimation of actual cross sectional area of the capillary tubing with the image of calibrated scanning electron microscopy. Within the given concentration range, an excellent measurement linearity (R2 = 0.999) was achieved with appropriate data processing for the correction of the events of double molecules (detection of double molecules opposed to single molecule detection assumed, which occurs due to their coincidental passing of the detection zone). The validity of the proposed method was confirmed from the close agreement with the results of quantitation of enzymatically released nucleotides using capillary electrophoresis.

  12. A single standardized practical training for surgical scrubbing according to EN1500: Effect Quantification, value of the standardized method and comparison with clinical reference groups

    PubMed Central

    Fichtner, Andreas; Haupt, Elke; Karwath, Tobias; Wullenk, Katharina; Pöhlmann, Christoph; Jatzwauk, Lutz

    2013-01-01

    The standardized training of practical competences in skills labs is relatively new among German Medical Faculties. The broad acceptance and outstanding evaluation results do not provide objective data on the efficiency and cost-efficiency of these trainings. This study aims on the quantification of the teaching effect of the surgical scrubbing technique EN1500 and its comparison with clinical references of OR personnel. Methods: 161 4th year medical students were randomized into intervention and control group. The intervention group received a 45 minute standardized peer-teaching training of practical competences necessary in the OR including the scrubbing according to EN1500. Fluorescence dye was mixed in the disinfectant solution. After hand disinfection, standardized fotographs and semi-automated digital processing resulted in quantification of the insufficiently covered hand area. These results were compared with the control group that received the training after the test. In order to provide information on the achieved clinical competence level, the results were compared with the two clinical reference groups. Results: The intervention group remained with 4,99% (SD 2,34) insufficiently covered hand area after the training compared to the control group 7,33% (SD 3,91), p<0,01. There was no significant difference between control group and reference groups: surgeons 9,32% (SD 4,97), scrub nurses 8,46% (SD 4,66). The student intervention group showed results that were significantly better than the clinical references. The methodic mistake remained negligible. In the sub-group analysis, the students with low or medium experience in surgical scrubbing and hand disinfection derived highest benefit from the training, whereas students with no or high experience did benefit less. All participants showed better results on hand palms compared to back of hand areas. Discussion: A single standardized peer-teaching of surgical scrubbing and hand disinfection according to EN

  13. Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes

    PubMed Central

    Schaeck, M.; De Spiegelaere, W.; De Craene, J.; Van den Broeck, W.; De Spiegeleer, B.; Burvenich, C.; Haesebrouck, F.; Decostere, A.

    2016-01-01

    The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3′/5′ integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof. PMID:26883391

  14. Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes.

    PubMed

    Schaeck, M; De Spiegelaere, W; De Craene, J; Van den Broeck, W; De Spiegeleer, B; Burvenich, C; Haesebrouck, F; Decostere, A

    2016-02-17

    The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3'/5' integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof.

  15. Validated comprehensive analytical method for quantification of coenzyme A activated compounds in biological tissues by online solid-phase extraction LC/MS/MS.

    PubMed

    Magnes, Christoph; Suppan, Maria; Pieber, Thomas R; Moustafa, Tarek; Trauner, Michael; Haemmerle, Guenter; Sinner, Frank M

    2008-08-01

    We report a robust, reliable, and comprehensive analytical method for the identification and quantification of the entire class of coenzyme A (CoA) activated substances, particularly short-, medium-, and long-chain acyl-CoAs derived from various biological tissues. This online SPE-LC/MS/MS-based method is characterized by a simple three-step sample preparation: (1) addition of buffer, organic solvents, and internal standards; (2) homogenization; and (3) centrifugation. The supernatant is injected directly into the SPE-LC/MS/MS system. Identification of CoA activated compounds is performed by accurate mass determination within the HPLC run. Method validation for short-, medium-, and long-chain acyl-CoA fatty acids revealed excellent quality. Accuracy was found to be between 87 and 107% and precision was between 0.1 and 12.8% in mouse skeletal muscle. The lower limit of quantification for all investigated compounds was well below 3.1% of estimated physiological levels in 200 mg of mouse tissue. Comparable results were obtained for mouse liver, mouse brown white adipose tissue and rat liver. For all investigated tissues, no matrix effect was observed.

  16. Preparation and analysis of a frozen mussel tissue reference material for the determination of trace organic constituents

    SciTech Connect

    Wise, S.A.; Benner, B.A. Jr.; Christensen, R.G.; Koster, B.J.; Kurz, J.; Schantz, M.M.; Zeisler, R. )

    1991-10-01

    A new mussel tissue Standard Reference Material (SRM) has been prepared and analyzed for trace organic and inorganic constituents. SRM 1974 (Organics in Mussel Tissue (Mytilus edulis)) is a frozen mussel tissue homogenate that has been certified for the concentrations of nine polycyclic aromatic hydrocarbons (PAHs) from results obtained from gas chromatography-mass spectrometry and reversed-phase liquid chromatography with fluorescence detection. Noncertified concentrations for 19 additional PAHs are also reported. Gas chromatography with electron capture detection and gas chromatography with mass spectrometric detection were used to provide noncertified concentrations for 13 polychlorinated biphenyl congeners and 9 chlorinated pesticides. In addition to the organic contaminants, noncertified concentrations for 36 trace elements were determined primarily by instrumental neutron activation analysis. SRM 1974 is the first frozen tissue SRM for environmental measurements of organic and inorganic constituents.

  17. A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

    PubMed

    White, H; Deprez, L; Corbisier, P; Hall, V; Lin, F; Mazoua, S; Trapmann, S; Aggerholm, A; Andrikovics, H; Akiki, S; Barbany, G; Boeckx, N; Bench, A; Catherwood, M; Cayuela, J-M; Chudleigh, S; Clench, T; Colomer, D; Daraio, F; Dulucq, S; Farrugia, J; Fletcher, L; Foroni, L; Ganderton, R; Gerrard, G; Gineikienė, E; Hayette, S; El Housni, H; Izzo, B; Jansson, M; Johnels, P; Jurcek, T; Kairisto, V; Kizilors, A; Kim, D-W; Lange, T; Lion, T; Polakova, K M; Martinelli, G; McCarron, S; Merle, P A; Milner, B; Mitterbauer-Hohendanner, G; Nagar, M; Nickless, G; Nomdedéu, J; Nymoen, D A; Leibundgut, E O; Ozbek, U; Pajič, T; Pfeifer, H; Preudhomme, C; Raudsepp, K; Romeo, G; Sacha, T; Talmaci, R; Touloumenidou, T; Van der Velden, V H J; Waits, P; Wang, L; Wilkinson, E; Wilson, G; Wren, D; Zadro, R; Ziermann, J; Zoi, K; Müller, M C; Hochhaus, A; Schimmel, H; Cross, N C P; Emons, H

    2015-02-01

    Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

  18. SPM analysis of parametric (R)-[11C]PK11195 binding images: plasma input versus reference tissue parametric methods.

    PubMed

    Schuitemaker, Alie; van Berckel, Bart N M; Kropholler, Marc A; Veltman, Dick J; Scheltens, Philip; Jonker, Cees; Lammertsma, Adriaan A; Boellaard, Ronald

    2007-05-01

    (R)-[11C]PK11195 has been used for quantifying cerebral microglial activation in vivo. In previous studies, both plasma input and reference tissue methods have been used, usually in combination with a region of interest (ROI) approach. Definition of ROIs, however, can be labourious and prone to interobserver variation. In addition, results are only obtained for predefined areas and (unexpected) signals in undefined areas may be missed. On the other hand, standard pharmacokinetic models are too sensitive to noise to calculate (R)-[11C]PK11195 binding on a voxel-by-voxel basis. Linearised versions of both plasma input and reference tissue models have been described, and these are more suitable for parametric imaging. The purpose of this study was to compare the performance of these plasma input and reference tissue parametric methods on the outcome of statistical parametric mapping (SPM) analysis of (R)-[11C]PK11195 binding. Dynamic (R)-[11C]PK11195 PET scans with arterial blood sampling were performed in 7 younger and 11 elderly healthy subjects. Parametric images of volume of distribution (Vd) and binding potential (BP) were generated using linearised versions of plasma input (Logan) and reference tissue (Reference Parametric Mapping) models. Images were compared at the group level using SPM with a two-sample t-test per voxel, both with and without proportional scaling. Parametric BP images without scaling provided the most sensitive framework for determining differences in (R)-[11C]PK11195 binding between younger and elderly subjects. Vd images could only demonstrate differences in (R)-[11C]PK11195 binding when analysed with proportional scaling due to intersubject variation in K1/k2 (blood-brain barrier transport and non-specific binding).

  19. X-ray microtomography (micro-CT): a reference technology for high-resolution quantification of xylem embolism in trees.

    PubMed

    Cochard, H; Delzon, S; Badel, E

    2015-01-01

    As current methods for measuring xylem embolism in trees are indirect and prone to artefacts, there is an ongoing controversy over the capacity of trees to resist or recover from embolism. The debate will not end until we get direct visualization of the vessel content. Here, we propose desktop X-ray microtomography (micro-CT) as a reference direct technique to quantify xylem embolism and thus validate more widespread measurements based upon either hydraulic or acoustic methods. We used desktop micro-CT to measure embolism levels in dehydrated or centrifuged shoots of laurel - a long-vesseled species thought to display daily cycles of embolism formation and refilling. Our direct observations demonstrate that this Mediterranean species is highly resistant to embolism and is not vulnerable to drought-induced embolism in a normal range of xylem tensions. We therefore recommend that embolism studies in long-vesseled species should be validated by direct methods such as micro-CT to clear up any misunderstandings on their physiology.

  20. Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax).

    PubMed

    Mitter, Karin; Kotoulas, Georgios; Magoulas, Antonios; Mulero, Victor; Sepulcre, Pilar; Figueras, Antonio; Novoa, Beatrice; Sarropoulou, Elena

    2009-08-01

    The expression level of mRNA can vary significantly in different experimental conditions, such as stress, infection, developmental stage or tissue. Suitable reference genes are expected to exhibit constant expression levels. However no single gene is constitutively expressed in all cell types and under all experimental conditions. It has become clear that expression stability of the intended reference gene has to be examined before each experiment. For expression studies using quantitative real-time PCR (qPCR) at least two reference genes have to be applied. So far expression studies in the European seabass (Dicentrarchus labrax) as well as in the Gilthead seabream (Sparus aurata) have been performed with only one reference gene (S18, Ef-1 alpha or Gapdh). Though significant variations showed up in other teleost species such as the Atlantic halibut and the zebrafish affirming the need for proper normalization strategies, the present study aims at identifying suitable reference genes among nine candidates [glyceraldehyde-phosphate-dehydrogenase (Gapdh), beta-actin (two regions of beta-actin), 40S ribosomal protein S30 (Fau), ribosomal protein L13 a (L13a), beta2-tubulin (Tubb2) and tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein (Tyr)] for expression analysis of 8 developmental stages and a tissue panel (spleen, liver, kidney and brain) with samples infected with Nodavirus and Vibrio anguillarum in D. labrax. Besides the analysis of raw Ct-values, the gene expression stability was determined using two different software applications BestKeeper and NormFinder. According to both algorithms the best two reference genes for an appropriate normalization approach during D. labrax development are Ef-1 alpha and L13a whereas in the tissue panel Fau and L13a are recommended for qPCR normalization.

  1. Selection of Reference Genes for Gene Expression Normalization in Peucedanum praeruptorum Dunn under Abiotic Stresses, Hormone Treatments and Different Tissues.

    PubMed

    Zhao, Yucheng; Luo, Jun; Xu, Sheng; Wang, Wei; Liu, Tingting; Han, Chao; Chen, Yijun; Kong, Lingyi

    2016-01-01

    Peucedanum praeruptorum Dunn is one of the main traditional Chinese medicines producing coumarins and plenty of literatures are focused on the biosynthesis of coumarins. Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used method in studying the biosynthesis pathway and the selection of reference genes plays a crucial role in accurate normalization. To facilitate biosynthesis study of coumarins, twelve candidate reference genes were selected from the transcriptome database of P. praeruptorum according to previous studies. Then, BestKeeper, geNoFrm and NormFinder were used for selecting stably expressed reference genes in different tissues and under various stress treatments. The results indicated that, among the twelve candidate reference genes, the SAND family protein (SAND), actin 2 (ACT2), ubiquitin-conjugating enzyme 9 (UBC9), protein phosphatase 2A gene (PP2A) and polypyrimidine tract-binding protein (PTBP1) were the most stable reference genes under different experimental treatments, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin beta-6 (TUB6) were the least stable genes. In addition, the suitability of SAND, TIP41-like protein (TIP41), UBC9, ACT2, TUB6 and their combination as reference genes were confirmed by normalizing the expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in different treatments. This work is the first survey of the stability of reference genes in P. praeruptorum and provides guidelines to obtain more accurate qRT-PCR results in P. praeruptorum and other plant species.

  2. Selection of Reference Genes for Gene Expression Normalization in Peucedanum praeruptorum Dunn under Abiotic Stresses, Hormone Treatments and Different Tissues

    PubMed Central

    Zhao, Yucheng; Luo, Jun; Xu, Sheng; Wang, Wei; Liu, Tingting; Han, Chao; Chen, Yijun; Kong, Lingyi

    2016-01-01

    Peucedanum praeruptorum Dunn is one of the main traditional Chinese medicines producing coumarins and plenty of literatures are focused on the biosynthesis of coumarins. Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used method in studying the biosynthesis pathway and the selection of reference genes plays a crucial role in accurate normalization. To facilitate biosynthesis study of coumarins, twelve candidate reference genes were selected from the transcriptome database of P. praeruptorum according to previous studies. Then, BestKeeper, geNoFrm and NormFinder were used for selecting stably expressed reference genes in different tissues and under various stress treatments. The results indicated that, among the twelve candidate reference genes, the SAND family protein (SAND), actin 2 (ACT2), ubiquitin-conjugating enzyme 9 (UBC9), protein phosphatase 2A gene (PP2A) and polypyrimidine tract-binding protein (PTBP1) were the most stable reference genes under different experimental treatments, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin beta-6 (TUB6) were the least stable genes. In addition, the suitability of SAND, TIP41-like protein (TIP41), UBC9, ACT2, TUB6 and their combination as reference genes were confirmed by normalizing the expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in different treatments. This work is the first survey of the stability of reference genes in P. praeruptorum and provides guidelines to obtain more accurate qRT-PCR results in P. praeruptorum and other plant species. PMID:27022972

  3. Reference Tissue-Based Kinetic Evaluation of 18F-AV-1451 for Tau Imaging.

    PubMed

    Baker, Suzanne L; Lockhart, Samuel N; Price, Julie C; He, Mark; Huesman, Ronald H; Schonhaut, Daniel; Faria, Jamie; Rabinovici, Gil; Jagust, William J

    2017-02-01

    The goal of this paper was to evaluate the in vivo kinetics of the novel tau-specific PET radioligand (18)F-AV-1451 in cognitively healthy control (HC) and Alzheimer disease (AD) subjects, using reference region analyses.

  4. Dual energy computed tomography for quantification of tissue urate deposits in tophaceous gout: help from modern physics in the management of an ancient disease.

    PubMed

    Bacani, A Kirstin; McCollough, Cynthia H; Glazebrook, Katrina N; Bond, Jeffrey R; Michet, Clement J; Milks, Jeffrey; Manek, Nisha J

    2012-01-01

    Gout has been recognized for centuries but is also a modern day scourge. It is the most common type of inflammatory arthritis in men and appears to be increasing in both incidence and prevalence (Arromdee et al. in J Rheumatol 29(11):2403-2406, 2002). Despite these facts, few advances have been made in the diagnosis and treatment of gout for over 50 years. Difficult cases of gout challenge available therapeutic options. It is only recently that the Food and Drug Administration has approved febuxostat as a treatment option for patients intolerant of allopurinol. We describe a difficult case of tophaceous gout notable for several reasons: utilization of rasburicase as uricolytic treatment to dramatically reduce tissue urate burden; treatment of gout flares with interleukin-1β inhibition; and quantification of tissue urate with novel dual energy computed tomography technology before and after uricolytic therapy.

  5. Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid, glutamine, pyroglutamic acid, GABA and theanine in mouse brain tissues.

    PubMed

    Inoue, Koichi; Miyazaki, Yasuto; Unno, Keiko; Min, Jun Zhe; Todoroki, Kenichiro; Toyo'oka, Toshimasa

    2016-01-01

    In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r(2)  > 0.999, range from 10 pmol/mL to 50 mol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus.

  6. NOISE 2 imaging system: seeing through scattering tissue with a reference point

    NASA Astrophysics Data System (ADS)

    Abookasis, David; Rosen, Joseph

    2004-05-01

    We propose a fly-eye-like imaging system for seeing objects embedded in scattering media. Objects are recovered from many speckled images observed by a digital camera through a microlens array. Each microlens in the array generates a speckle image of the object buried between two layers of chicken breast tissue. In the computer each image is Fourier transformed jointly with an image of the speckled pointlike source captured under the same conditions. A set of the squared magnitudes of the Fourier-transformed pictures is accumulated to form a single average picture. This final picture is again Fourier transformed, resulting in the reconstruction of the hidden object.

  7. UPLC-MS method for quantification of pterostilbene and its application to comparative study of bioavailability and tissue distribution in normal and Lewis lung carcinoma bearing mice.

    PubMed

    Deng, Li; Li, Yongzhi; Zhang, Xinshi; Chen, Bo; Deng, Yulin; Li, Yujuan

    2015-10-10

    A UPLC-MS method was developed for determination of pterostilbene (PTS) in plasma and tissues of mice. PTS was separated on Agilent Zorbax XDB-C18 column (50 × 2.1 mm, 1.8 μm) with gradient mobile phase at the flow rate of 0.2 ml/min. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode. The linear calibration curve of PTS in mouse plasma and tissues ranged from 1.0 to 5000 and 0.50 to 500 ng/ml (r(2)>0.9979), respectively, with lowest limits of quantification (LLOQ) were between 0.5 and 2.0 ng/ml, respectively. The accuracy and precision of the assay were satisfactory. The validated method was applied to the study of bioavailability and tissue distribution of PTS in normal and Lewis lung carcinoma (LLC) bearing mice. The bioavailability of PTS (dose 14, 28 and 56 mg/kg) in normal mice were 11.9%, 13.9% and 26.4%, respectively; and the maximum level (82.1 ± 14.2 μg/g) was found in stomach (dose 28 mg/kg). The bioavailability, peak concentration (Cmax), time to peak concentration (Tmax) of PTS in LLC mice was increased compared with normal mice. The results indicated the UPLC-MS method is reliable and bioavailability and tissue distribution of PTS in normal and LLC mice were dramatically different.

  8. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve

    PubMed Central

    Rueda-Martínez, Carmen; Fernández, M. Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

    2016-01-01

    Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180–240 days old) and 56 old (300–440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta. PMID:27711171

  9. A new formalism for the quantification of tissue perfusion by the destruction-replenishment method in contrast ultrasound imaging.

    PubMed

    Arditi, Marcel; Frinking, Peter J A; Zhou, Xiang; Rognin, Nicolas G

    2006-06-01

    A new formalism is presented for the destruction-replenishment perfusion quantification approach at low mechanical index. On the basis of physical considerations, best-fit methods should be applied using perfusion functions with S-shape characteristics. These functions are first described for the case of a geometry with a single flow velocity, then extended to the case of vascular beds with blood vessels having multiple flow velocity values and directions. The principles guiding the analysis are, on one hand, a linearization of video echo signals to overcome the log-compression of the imaging instrument, and, on the other hand, the spatial distribution of the transmit-receive ultrasound beam in the elevation direction. An in vitro model also is described; it was used to confirm experimentally the validity of the approach using a commercial contrast agent. The approach was implemented in the form of a computer program, taking as input a sequence of contrast-specific images, as well as parameters related to the ultrasound imaging equipment used. The generated output is either flow-parameter values computed in regions-of-interest, or parametric flow-images (e.g., mean velocity, mean transit time, mean flow, flow variance, or skewness). This approach thus establishes a base for extracting information about the morphology of vascular beds in vivo, and could allow absolute quantification provided that appropriate instrument calibration is implemented.

  10. Automated quantification of adipose and skeletal muscle tissue in whole-body MRI data for epidemiological studies

    NASA Astrophysics Data System (ADS)

    Wald, Diana; Teucher, Birgit; Dinkel, Julien; Kaaks, Rudolf; Delorme, Stefan; Meinzer, Hans-Peter; Heimann, Tobias

    2012-03-01

    The ratio between the amount of adipose and skeletal muscle tissue is an important determinant of metabolic health. Recent developments in MRI technology allow whole body scans to be performed for accurate assessment of body composition. In the present study, a total of 194 participants underwent a 2-point Dixon MRI sequence of the whole body. A fully automated image segmentation method quantifies the amount of adipose and skeletal muscle tissue by applying standard image processing techniques including thresholding, region growing and morphological operators. The adipose tissue is further divided into subcutaneous and visceral adipose tissue by using statistical shape models. All images were visually inspected. The quantitative analysis was performed on 44 whole-body MRI data using manual segmentations as ground truth data. We achieved 3.3% and 6.3% of relative volume difference between the manual and automated segmentation of subcutaneous and visceral adipose tissue, respectively. The validation of skeletal muscle tissue segmentation resulted in a relative volume difference of 7.8 +/- 4.2% and a volumetric overlap error of 6.4 +/- 2.3 %. To our knowledge, we are first to present a fully automated method which quantifies adipose and skeletal muscle tissue in whole-body MRI data. Due to the fully automated approach, results are deterministic and free of user bias. Hence, the software can be used in large epidemiological studies for assessing body fat distribution and the ratio of adipose to skeletal muscle tissue in relation to metabolic disease risk.

  11. High resolution systematic digital histological quantification of cardiac fibrosis and adipose tissue in phospholamban p.Arg14del mutation associated cardiomyopathy.

    PubMed

    Gho, Johannes M I H; van Es, René; Stathonikos, Nikolas; Harakalova, Magdalena; te Rijdt, Wouter P; Suurmeijer, Albert J H; van der Heijden, Jeroen F; de Jonge, Nicolaas; Chamuleau, Steven A J; de Weger, Roel A; Asselbergs, Folkert W; Vink, Aryan

    2014-01-01

    Myocardial fibrosis can lead to heart failure and act as a substrate for cardiac arrhythmias. In dilated cardiomyopathy diffuse interstitial reactive fibrosis can be observed, whereas arrhythmogenic cardiomyopathy is characterized by fibrofatty replacement in predominantly the right ventricle. The p.Arg14del mutation in the phospholamban (PLN) gene has been associated with dilated cardiomyopathy and recently also with arrhythmogenic cardiomyopathy. Aim of the present study is to determine the exact pattern of fibrosis and fatty replacement in PLN p.Arg14del mutation positive patients, with a novel method for high resolution systematic digital histological quantification of fibrosis and fatty tissue in cardiac tissue. Transversal mid-ventricular slices (n = 8) from whole hearts were collected from patients with the PLN p.Arg14del mutation (age 48±16 years; 4 (50%) male). An in-house developed open source MATLAB script was used for digital analysis of Masson's trichrome stained slides (http://sourceforge.net/projects/fibroquant/). Slides were divided into trabecular, inner and outer compact myocardium. Per region the percentage of connective tissue, cardiomyocytes and fatty tissue was quantified. In PLN p.Arg14del mutation associated cardiomyopathy, myocardial fibrosis is predominantly present in the left posterolateral wall and to a lesser extent in the right ventricular wall, whereas fatty changes are more pronounced in the right ventricular wall. No difference in distribution pattern of fibrosis and adipocytes was observed between patients with a clinical predominantly dilated and arrhythmogenic cardiomyopathy phenotype. In the future, this novel method for quantifying fibrosis and fatty tissue can be used to assess cardiac fibrosis and fatty tissue in animal models and a broad range of human cardiomyopathies.

  12. Quantification of Arachidonic Acid and Its Metabolites in Rat Tissues by UHPLC-MS/MS: Application for the Identification of Potential Biomarkers of Benign Prostatic Hyperplasia

    PubMed Central

    Bian, Qiaoxia; Wang, Weihui; Wang, Nannan; Peng, Yan; Ma, Wen; Dai, Ronghua

    2016-01-01

    To evaluate the potential relationship between benign prostatic hyperplasia (BPH) and the arachidonic acid (AA) metabolome, a UHPLC—MS/MS method has been developed and validated for simultaneous determination of AA and its cyclooxygenase(COX) and lipoxygenase(LOX) pathway metabolites (15-HETE, 12-HETE, TXA2, 5-HETE, AA, PGI2, PGF2α, 8-HETE, PGD2, PGE2 and LTB4) in rat tissues. The analytes were extracted from tissue samples with a protein precipitation procedure and then separated on a Shim-pack XR-ODSC18 column with 0.05% formic acid in water (pH adjusted with dilute ammonia) and methanol:acetonitrile (20:80, v/v). Detection was performed on a UHPLC—MS/MS system with electrospray negative ionization (ESI) and a multiple reaction-monitoring mode. The lower limits of quantification (LLOQ) were 0.25–50 ng/mL for all of the analytes in the prostate, seminal, bladder, liver and kidney tissues. The absolute recoveries of the analytes from all of the tissues were more than 50%. By means of the method developed, the AA metabolites in tissue samples from Sham and BPH group rats were determined. The eleven biomarkers in the BPH group prostate, seminal, bladder, liver and kidney tissues were significantly higher than those of the sham group, indicating that BPH fortified the inducible expression of COX and LOX, as well as increased the production of AA and eicosanoids. The method described here offers a useful tool for the evaluation of complex regulatory eicosanoids responses in vivo. PMID:27893755

  13. Optimization of a gas chromatography-mass spectrometry method with methyl chloroformate derivatization for quantification of amino acids in plant tissue.

    PubMed

    Vancompernolle, Bram; Croes, Kim; Angenon, Geert

    2016-04-01

    Rapid, easy and reliable quantification of amino acids is crucial in research on plant amino acid metabolism and nutritional improvement of crops via enrichment of essential amino acids. A recently reported analysis method, based on solid phase extraction (SPE), derivatization with methyl chloroformate and gas chromatography-mass spectrometry was optimized and tested on three-week-old Arabidopsis thaliana leaf tissues. Optimization of the SPE cleanup yielded recovery rates of minimum 95% for all amino acids (except arginine). Variations in accuracy and precision did not exceed 12.5%, except for cysteine, histidine and tryptophane, which were excluded from analysis. Quantification of overlapping peaks for isoleucine/threonine and proline/asparagine was possible by selection of two specific fragment ions for each amino acid. Of the 16 selected amino acids, 14 were quantified successfully in at least 75% of the samples, while methionine and tyrosine were only quantifiable in 6% and 42%, respectively. A case study on the aspartate super pathway confirmed the applicability of the optimized method on wild type and genetically modified plants: external supplementation of methionine or lysine yielded a 146-fold or 27-fold increase in the respective absolute amino acid levels compared with the control treatment. Induced expression of dhdps-r1 (a mutated lysine biosynthesis gene encoding a feedback insensitive enzyme) caused an 83-fold increase in absolute lysine levels.

  14. Quantification of vitamin D and 25-hydroxyvitamin D in soft tissues by liquid chromatography-tandem mass spectrometry.

    PubMed

    Lipkie, Tristan E; Janasch, Amber; Cooper, Bruce R; Hohman, Emily E; Weaver, Connie M; Ferruzzi, Mario G

    2013-08-01

    Inadequate data on tissue distribution of vitamin D and its metabolites remains a barrier to defining health outcomes of vitamin D intake and 25-hydroxyvitamin D (25(OH)D) status. The purpose of this study was to develop a method for the analysis of vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), 25(OH)D2, and 25(OH)D3 in soft tissues, and determine distribution in select tissues from a dose-response study of vitamin D2 and vitamin D3 in rats. Liver, gastrocnemius muscle, and epididymal fat homogenates were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization following liquid-liquid extraction, solid-phase extraction, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). A dose-response was observed in most tissues for vitamin D and 25(OH)D from both vitamers. Vitamin D concentration was greater in epididymal fat than gastrocnemius muscle and liver, but 25(OH)D concentration was not significantly different between tissues. Soft tissues of rats fed crystalline vitamin D3 had higher concentrations of total vitamin D than those of rats fed yeast-derived vitamin D2, while total 25(OH)D concentrations were similar between vitamin D sources. This method is well suited to more complete studies of vitamin D bioavailability and metabolite tissue distribution.

  15. Quantification of vitamin D and 25-hydroxyvitamin D in soft tissues by liquid chromatography—tandem mass spectrometry

    PubMed Central

    Lipkie, Tristan E.; Janasch, Amber; Cooper, Bruce R.; Hohman, Emily E.; Weaver, Connie M.; Ferruzzi, Mario G.

    2013-01-01

    Inadequate data on tissue distribution of vitamin D and its metabolites remains a barrier to defining health outcomes of vitamin D intake and 25-hydroxyvitamin D (25(OH)D) status. The purpose of this study was to develop a method for the analysis of vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), 25(OH)D2, and 25(OH)D3 in soft tissues, and determine distribution in select tissues from a dose-response study of vitamin D2 and vitamin D3 in rats. Liver, gastrocnemius muscle, and epididymal fat homogenates were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization following liquid-liquid extraction, solid-phase extraction, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). A dose-response was observed in most tissues for vitamin D and 25(OH)D from both vitamers. Vitamin D concentration was greater in epididymal fat than gastrocnemius muscle and liver, but 25(OH)D concentration was not significantly different between tissues. Soft tissues of rats fed crystalline vitamin D3 had higher concentrations of total vitamin D than those of rats fed yeast-derived vitamin D2, while total 25(OH)D concentrations were similar between vitamin D sources. This method is well suited to more complete studies of vitamin D bioavailability and metabolite tissue distribution. PMID:23811497

  16. Tissue-specific Calibration of Real-time PCR Facilitates Absolute Quantification of Plasmid DNA in Biodistribution Studies

    PubMed Central

    Ho, Joan K; White, Paul J; Pouton, Colin W

    2016-01-01

    Analysis of the tissue distribution of plasmid DNA after administration of nonviral gene delivery systems is best accomplished using quantitative real-time polymerase chain reaction (qPCR), although published strategies do not allow determination of the absolute mass of plasmid delivered to different tissues. Generally, data is expressed as the mass of plasmid relative to the mass of genomic DNA (gDNA) in the sample. This strategy is adequate for comparisons of efficiency of delivery to a single site but it does not allow direct comparison of delivery to multiple tissues, as the mass of gDNA extracted per unit mass of each tissue is different. We show here that by constructing qPCR standard curves for each tissue it is possible to determine the dose of intact plasmid remaining in each tissue, which is a more useful parameter when comparing the fates of different formulations of DNA. We exemplify the use of this tissue-specific qPCR method by comparing the delivery of naked DNA, cationic DNA complexes, and neutral PEGylated DNA complexes after intramuscular injection. Generally, larger masses of intact plasmid were present 24 hours after injection of DNA complexes, and neutral complexes resulted in delivery of a larger mass of intact plasmid to the spleen. PMID:27701400

  17. Calibration-free absolute quantification of optical absorption coefficients using acoustic spectra in 3D photoacoustic microscopy of biological tissue.

    PubMed

    Guo, Zijian; Hu, Song; Wang, Lihong V

    2010-06-15

    Optical absorption is closely associated with many physiological important parameters, such as the concentration and oxygen saturation of hemoglobin, and it can be used to quantify the concentrations of nonfluorescent molecules. We propose a method to use acoustic spectra of photoacoustic signals to quantify the absolute optical absorption. This method is self-calibrating and thus insensitive to variations in the optical fluence. Factors such as system bandwidth and acoustic attenuation can affect the quantification but can be canceled by dividing the acoustic spectra measured at two optical wavelengths. Using optical-resolution photoacoustic microscopy, we quantified the absolute optical absorption of black ink samples with various concentrations. We also quantified both the concentration and oxygen saturation of hemoglobin in a live mouse in absolute units.

  18. Localisation and quantification of benzalkonium chloride in eye tissue by TOF-SIMS imaging and liquid chromatography mass spectrometry.

    PubMed

    Desbenoit, Nicolas; Schmitz-Afonso, Isabelle; Baudouin, Christophe; Laprévote, Olivier; Touboul, David; Brignole-Baudouin, Françoise; Brunelle, Alain

    2013-05-01

    Benzalkonium (BAK) chloride is the most commonly used preservative in eye drops. It is generally composed of benzyldimethyldodecylammonium C12 and benzyldimethyltetradecylammonium C14 and is supposed to increase penetration of active compounds. However, numerous studies have reported its toxic effect to ocular surface especially in long-term treatments like against glaucoma, a sight-threatening disease. Albino rabbits were treated with a hyperosmolar solution and a high concentration of BAK solution for 1 month. Enucleated eyes were cryo-sectioned and analysed by mass spectrometry. Mass spectrometry imaging using time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to characterize the spatial distribution and to determine the relative quantity of BAK at the surface of rabbit eye sections. Liquid chromatography coupled with mass spectrometry (LC-MS) using a hybrid linear ion trap-Orbitrap® mass spectrometer was used to obtain relative quantification of BAK at the sample surface. TOF-SIMS images of BAK ions indicated a distribution at the ocular surface and in deeper structures. Didecyldimethylammonium (DDMAC), which is used in hospitals as a substitute for BAK, was also detected and showed an accumulation around the eyes. After extraction with acetonitrile and chromatographic separation using a Gemini C18 column and an original elution gradient, the relative quantities of BAK and DDMAC present in the whole eye section surface were determined. This LC-MS method was validated in terms of limits of quantification, linearity, repeatability and reproducibility and its feasibility was evaluated in surgically obtained human samples. Specimens of iris, lens capsule or trabecular meshwork were found with significant levels of BAK and DDMAC, thus confirming the penetration of BAK in deep ocular structures, with potential deleterious effects induced by this cytotoxic compound. The analytical method developed here could therefore be of primary interest in

  19. Long-term stability and temporal trends of organic contaminants in four collections of mussel tissue frozen standard reference materials.

    PubMed

    Schantz, Michele M; Pugh, Rebecca S; Pol, Stacy S Vander; Wise, Stephen A

    2015-04-01

    The stability of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and chlorinated pesticides in frozen mussel tissue Standard Reference Materials (SRMs) stored at -80 °C was assessed by analyzing samples of SRM 1974, SRM 1974a, and SRM 1974b Organics in Mussel Tissue (Mytilus edulis) periodically over 25 y, 20 y, and 12 y, respectively. The most recent analyses were performed during the certification of the fourth release of this material, SRM 1974c. Results indicate the concentrations of these persistent organic pollutants have not changed during storage at -80 °C. In addition, brominated diphenyl ethers (BDEs) were quantified in each of the materials during this study. The stability information is important for on-going monitoring studies collecting large quantities of samples for future analyses (i.e., formally established specimen banking programs). Since all four mussel tissue SRMs were prepared from mussels collected at the same site in Dorchester Bay, MA, USA, the results provide a temporal trend study for these contaminants over a 17 year period (1987 to 2004).

  20. Selection of reliable reference genes for normalization of quantitative RT-PCR from different developmental stages and tissues in amphioxus

    PubMed Central

    Zhang, Qi-Lin; Zhu, Qian-Hua; Liao, Xin; Wang, Xiu-Qiang; Chen, Tao; Xu, Han-Ting; Wang, Juan; Yuan, Ming-Long; Chen, Jun-Yuan

    2016-01-01

    Amphioxus is a closest living proxy to the ancestor of cephalochordates with vertebrates, and key animal for novel understanding in the evolutionary origin of vertebrate body plan, genome, tissues and immune system. Reliable analyses using quantitative real-time PCR (qRT-PCR) for answering these scientific questions is heavily dependent on reliable reference genes (RGs). In this study, we evaluated stability of thirteen candidate RGs in qRT-PCR for different developmental stages and tissues of amphioxus by four independent (geNorm, NormFinder, BestKeeper and deltaCt) and one comparative algorithms (RefFinder). The results showed that the top two stable RGs were the following: (1) S20 and 18 S in thirteen developmental stages, (2) EF1A and ACT in seven normal tissues, (3) S20 and L13 in both intestine and hepatic caecum challenged with lipopolysaccharide (LPS), and (4) S20 and EF1A in gill challenged with LPS. The expression profiles of two target genes (EYA and HHEX) in thirteen developmental stages were used to confirm the reliability of chosen RGs. This study identified optimal RGs that can be used to accurately measure gene expression under these conditions, which will benefit evolutionary and functional genomics studies in amphioxus. PMID:27869224

  1. Abdominal adipose tissue quantification on water-suppressed and non-water-suppressed MRI at 3T using semi-automated FCM clustering algorithm

    NASA Astrophysics Data System (ADS)

    Valaparla, Sunil K.; Peng, Qi; Gao, Feng; Clarke, Geoffrey D.

    2014-03-01

    Accurate measurements of human body fat distribution are desirable because excessive body fat is associated with impaired insulin sensitivity, type 2 diabetes mellitus (T2DM) and cardiovascular disease. In this study, we hypothesized that the performance of water suppressed (WS) MRI is superior to non-water suppressed (NWS) MRI for volumetric assessment of abdominal subcutaneous (SAT), intramuscular (IMAT), visceral (VAT), and total (TAT) adipose tissues. We acquired T1-weighted images on a 3T MRI system (TIM Trio, Siemens), which was analyzed using semi-automated segmentation software that employs a fuzzy c-means (FCM) clustering algorithm. Sixteen contiguous axial slices, centered at the L4-L5 level of the abdomen, were acquired in eight T2DM subjects with water suppression (WS) and without (NWS). Histograms from WS images show improved separation of non-fatty tissue pixels from fatty tissue pixels, compared to NWS images. Paired t-tests of WS versus NWS showed a statistically significant lower volume of lipid in the WS images for VAT (145.3 cc less, p=0.006) and IMAT (305 cc less, p<0.001), but not SAT (14.1 cc more, NS). WS measurements of TAT also resulted in lower fat volumes (436.1 cc less, p=0.002). There is strong correlation between WS and NWS quantification methods for SAT measurements (r=0.999), but poorer correlation for VAT studies (r=0.845). These results suggest that NWS pulse sequences may overestimate adipose tissue volumes and that WS pulse sequences are more desirable due to the higher contrast generated between fatty and non-fatty tissues.

  2. Quantification of total mercury in liver and heart tissue of Harbor Seals (Phoca vitulina) from Alaska USA

    SciTech Connect

    Marino, Kady B.; Hoover-Miller, Anne; Conlon, Suzanne; Prewitt, Jill; O'Shea, Stephen K.

    2011-11-15

    This study quantified the Hg levels in the liver (n=98) and heart (n=43) tissues of Harbor Seals (Phoca vitulina) (n=102) harvested from Prince William Sound and Kodiak Island Alaska. Mercury tissue dry weight (dw) concentrations in the liver ranged from 1.7 to 393 ppm dw, and in the heart from 0.19 to 4.99 ppm dw. Results of this study indicate liver and heart tissues' Hg ppm dw concentrations significantly increase with age. Male Harbor Seals bioaccumulated Hg in both their liver and heart tissues at a significantly faster rate than females. The liver Hg bioaccumulation rates between the harvest locations Kodiak Island and Prince William Sound were not found to be significantly different. On adsorption Hg is transported throughout the Harbor Seal's body with the partition coefficient higher for the liver than the heart. No significant differences in the bio-distribution (liver:heart Hg ppm dw ratios (n=38)) values were found with respect to either age, sex or geographic harvest location. In this study the age at which Hg liver and heart bioaccumulation levels become significantly distinct in male and female Harbor Seals were identified through a Tukey's analysis. Of notably concern to human health was a male Harbor Seal's liver tissue harvested from Kodiak Island region. Mercury accumulation in this sample tissue was determined through a Q-test to be an outlier, having far higher Hg concentrarion (liver 392 Hg ppm dw) than the general population sampled. - Highlights: Black-Right-Pointing-Pointer Mercury accumulation in the liver and heart of seals exceed food safety guidelines. Black-Right-Pointing-Pointer Accumulation rate is greater in males than females with age. Black-Right-Pointing-Pointer Liver mercury accumulation is greater than in the heart tissues. Black-Right-Pointing-Pointer Mercury determination by USA EPA Method 7473 using thermal decomposition.

  3. Agouti revisited: transcript quantification of the ASIP gene in bovine tissues related to protein expression and localization.

    PubMed

    Albrecht, Elke; Komolka, Katrin; Kuzinski, Judith; Maak, Steffen

    2012-01-01

    Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species.

  4. Dystrophin quantification

    PubMed Central

    Anthony, Karen; Arechavala-Gomeza, Virginia; Taylor, Laura E.; Vulin, Adeline; Kaminoh, Yuuki; Torelli, Silvia; Feng, Lucy; Janghra, Narinder; Bonne, Gisèle; Beuvin, Maud; Barresi, Rita; Henderson, Matt; Laval, Steven; Lourbakos, Afrodite; Campion, Giles; Straub, Volker; Voit, Thomas; Sewry, Caroline A.; Morgan, Jennifer E.; Flanigan, Kevin M.

    2014-01-01

    Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval. PMID:25355828

  5. Analytical and numerical quantification and comparison of the local electric field in the tissue for different electrode configurations

    PubMed Central

    Čorović, Selma; Pavlin, Mojca; Miklavčič, Damijan

    2007-01-01

    Background Electrochemotherapy and gene electrotransfer are novel promising treatments employing locally applied high electric pulses to introduce chemotherapeutic drugs into tumor cells or genes into target cells based on the cell membrane electroporation. The main focus of this paper was to calculate analytically and numerically local electric field distribution inside the treated tissue in two dimensional (2D) models for different plate and needle electrode configurations and to compare the local electric field distribution to parameter U/d, which is widely used in electrochemotherapy and gene electrotransfer studies. We demonstrate the importance of evaluating the local electric field distribution in electrochemotherapy and gene electrotransfer. Methods We analytically and numerically analyze electric field distribution based on 2D models for electrodes and electrode configurations which are most widely used in electrochemotherapy and gene electrotransfer. Analytical calculations were performed by solving the Laplace equation and numerical calculations by means of finite element method in two dimensions. Results We determine the minimal and maximal E inside the target tissue as well as the maximal E over the entire treated tissue for the given electrode configurations. By comparing the local electric field distribution calculated for different electrode configurations to the ratio U/d, we show that the parameter U/d can differ significantly from the actual calculated values of the local electric field inside the treated tissue. By calculating the needed voltage to obtain E > U/d inside the target tissue, we showed that better electric field distribution can be obtained by increasing the number and changing the arrangement of the electrodes. Conclusion Based on our analytical and numerical models of the local electric field distribution we show that the applied voltage, configuration of the electrodes and electrode position need to be chosen specifically for

  6. Detection and Quantification of CWD Prions in Fixed Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion

    PubMed Central

    Hoover, Clare E.; Davenport, Kristen A.; Henderson, Davin M.; Pulscher, Laura A.; Mathiason, Candace K.; Zabel, Mark D.; Hoover, Edward A.

    2016-01-01

    Traditional diagnostic detection of chronic wasting disease (CWD) relies on immunodetection of misfolded CWD prion protein (PrPCWD) by western blotting, ELISA, or immunohistochemistry (IHC). These techniques require separate sample collections (frozen and fixed) which may result in discrepancies due to variation in prion tissue distribution and assay sensitivities that limit detection especially in early and subclinical infections. Here, we harness the power of real-time quaking induced conversion (RT-QuIC) to amplify, detect, and quantify prion amyloid seeding activity in fixed paraffin-embedded (FPE) tissue sections. We show that FPE RT-QuIC has greater detection sensitivity than IHC in tissues with low PrPCWD burdens, including those that are IHC-negative. We also employ amyloid formation kinetics to yield a semi-quantitative estimate of prion concentration in a given FPE tissue. We report that FPE RT-QuIC has the ability to enhance diagnostic and investigative detection of disease-associated PrPRES in prion, and potentially other, protein misfolding disease states. PMID:27157060

  7. Quantification of inflammation in colonic tissue sections and wound healing in-vitro with digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Bettenworth, Dominik; Lenz, Philipp; Krausewitz, Philipp; Brückner, Markus; Ketelhut, Steffi; von Bally, Gert; Domagk, Dirk; Kemper, Björn

    2013-06-01

    We show that the tissue refractive index, obtained from quantitative digital holographic microscopy (DHM) phase contrast images of unstained histological colonic sections, is directly related to the degree of inflammation in experimental colitis. In addition, it is demonstrated that quantitative DHM phase contrast is capable to quantify in-vitro wound healing assays.

  8. Agouti Revisited: Transcript Quantification of the ASIP Gene in Bovine Tissues Related to Protein Expression and Localization

    PubMed Central

    Albrecht, Elke; Komolka, Katrin; Kuzinski, Judith; Maak, Steffen

    2012-01-01

    Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species. PMID:22530003

  9. Quantification of fluorophore concentration in tissue-simulating media by fluorescence measurements with a single optical fiber.

    PubMed

    Diamond, Kevin R; Patterson, Michael S; Farrell, Thomas J

    2003-05-01

    Quantifying fluorescent compounds in turbid media such as tissue is made difficult by the effects of multiple scattering and absorption of the excitation and emission light. The approach that we used was to measure fluorescence using a single 200-microm optical fiber as both the illumination source and the detector. Fluorescence of aluminum phthalocyanine tetrasulfonate (AlPcS4) was measured over a wide range of fluorophore concentrations and optical properties in tissue-simulating phantoms. A root-mean-square accuracy of 10.6% in AlPcS4 concentration was attainable when fluorescence was measured either interstitially or at the phantom surface. The individual effects of scattering, absorption, and the scattering phase function on the fluorescence signal were also studied by experiments and Monte Carlo simulations.

  10. Evaluation of EDXRF configurations to improve the limit of detection and exposure for in vivo quantification of gadolinium in tumor tissue

    NASA Astrophysics Data System (ADS)

    Santibáñez, M.; Vásquez, M.; Figueroa, R. G.; Valente, M.

    2016-05-01

    In this paper the configuration of an Energy Dispersive X-Ray Fluorescence (EDXRF) system optimized for in vivo quantification of gadolinium in tumor tissue was studied. The system was configured using XMI-MSIM software designed to predict the XRF spectral response using Monte Carlo simulations. The studied setup is comprised of an X-ray tube, tuned to different voltages, and a copper filter system configured with variable thickness, which emits a spectrally narrow beam centered on the specific excitation energy. The values for the central energy excitation and the spectral width were adjusted to optimize the system, using like figures of merit: minimization of the limit of detection, measurement uncertainty and radiation exposure. These values were obtained in two stages. The first was successive simulations of incident spectra with central energy in the range of 50-70 keV. The second was comprised of simulations with incident spectra of different widths (8-29 keV), all with the same determined central energy, evaluating the limit of detection depending on the exposure. This made it possible to find the best balance between system sensitivity and the delivered dose. The obtained results were compared with those produced by radioactive sources of 241Am whose activity was set to produce the same exposure as the proposed setup. To evaluate the feasibility of in vivo quantification, a set of tumor phantoms of 1-6 cm3 at different depths and labeled with a gadolinium concentration of 250 ppm was evaluated. From the resulting spectrum, calibration curves were obtained in function of the size and depth of the tumor, allowing for the evaluation of the potential of the methodology.

  11. Development and validation of a high performance liquid chromatography quantification method of levo-tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study.

    PubMed

    Abdallah, Inas A; Huang, Peng; Liu, Jing; Lee, David Y; Liu-Chen, Lee-Yuan; Hassan, Hazem E

    2017-04-01

    Levo-tetrahydropalmatine (l-THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l-THP and its desmethyl metabolites l-corydalmine (l-CD) and l-corypalmine (l-CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid-liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed-phase Symmetry® C18 column (4.6 × 150 mm, 5 μm) at 25°C. The mobile phase consisted of acetonitrile-methanol-10 mm ammonium phosphate (pH 3) (10:30:60, v/v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1-10,000 ng/mL. The intra- and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l-THP in rats. Taken together, the developed method can be applied for bioanalysis of l-THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.

  12. PCR assay based on a microsatellite-containing locus for detection and quantification of Epichloë endophytes in grass tissue.

    PubMed Central

    Groppe, K; Boller, T

    1997-01-01

    A PCR assay which allows detection and quantification of Epichloë endophytes in tissues of the grass Bromus erectus is described. PCR with specific primers flanking a microsatellite-containing locus (MS primers) amplified fragments 300 to 400 bp in length from as little as 1.0 pg of fungal genomic DNA in 100 ng of DNA from infected plant material. When annealing temperatures were optimized, all Epichloë and Acremonium strains tested, representing many of the known taxonomic groups, yielded an amplification product, indicating that the MS primers may be useful for in planta detection of a variety of related species, including agronomically important Acremonium coenophialum and Acremonium lolii. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagnostic applications, a B. erectus-specific primer pair was designed for use in multiplex PCR to allow simultaneous amplification of plant and fungal DNA sequences, providing an internal control for PCR failure caused by inhibitory plant compounds present in DNA extracts. For quantitative applications, a heterologous control template in primer binding sites complementary to the MS primers was constructed for use in competitive PCR, allowing direct quantification of Epichloë in plant DNA extracts. The fungal DNA present in infected leaves of B. erectus between 1 and 20 pg per 100 ng of leaf DNA, but the amounts of fungal DNA present in the sheath and blade of a given leaf were correlated, indicating that the degree of infection varied between plant individuals but that leaves were colonized in a uniform way. PMID:9097449

  13. Computerized Automated Quantification of Subcutaneous and Visceral Adipose Tissue From Computed Tomography Scans: Development and Validation Study

    PubMed Central

    Kim, Young Jae; Park, Ji Won; Kim, Jong Wan; Park, Chan-Soo; Gonzalez, John Paul S; Lee, Seung Hyun

    2016-01-01

    Background Computed tomography (CT) is often viewed as one of the most accurate methods for measuring visceral adipose tissue (VAT). However, measuring VAT and subcutaneous adipose tissue (SAT) from CT is a time-consuming and tedious process. Thus, evaluating patients’ obesity levels during clinical trials using CT scans is both cumbersome and limiting. Objective To describe an image-processing-based and automated method for measuring adipose tissue in the entire abdominal region. Methods The method detects SAT and VAT levels using a separation mask based on muscles of the human body. The separation mask is the region that minimizes the unnecessary space between a closed path and muscle area. In addition, a correction mask, based on bones, corrects the error in VAT. Results To validate the method, the volume of total adipose tissue (TAT), SAT, and VAT were measured for a total of 100 CTs using the automated method, and the results compared with those from manual measurements obtained by 2 experts. Dice’s similarity coefficients (DSCs) between the first manual measurement and the automated result for TAT, SAT, and VAT are 0.99, 0.98, and 0.97, respectively. The DSCs between the second manual measurement and the automated result for TAT, SAT, and VAT are 0.98, 0.98, and 0.97, respectively. Moreover, intraclass correlation coefficients (ICCs) between the automated method and the results of the manual measurements indicate high reliability as the ICCs for the items are all .99 (P<.001). Conclusions The results described in this paper confirm the accuracy and reliability of the proposed method. The method is expected to be both convenient and useful in the clinical evaluation and study of obesity in patients who require SAT and VAT measurements. PMID:26846251

  14. Sources of variability in the quantification of tissue optical properties by multidiameter single-fiber reflectance and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Brooks, Sander; Hoy, Christopher L.; Amelink, Arjen; Robinson, Dominic J.; Nijsten, Tamar E.

    2015-05-01

    Recently, a multidiameter single-fiber reflectance and fluorescence spectroscopy device has been developed that enabled us to extract the autofluorescence of tissue that is corrected for the optical properties. Such a system has been incorporated in the population-based Rotterdam Study to investigate the autofluorescence of the skin. Since the device will be used by different operators over many years, it is essential that the results are comparable between users. It is, however, unclear how different methods of handling the probe might influence the outcome. Variability of blood oxygen saturation, blood volume fraction and vessel diameter, average gamma, reduced scattering coefficient at 800 nm, and integrated intrinsic fluorescence measured in three volunteers were assessed within and between eight untrained users. A variability of less than one standard deviation from the group mean was defined as an acceptable limit. Three mature volunteers were also included to assess the intrauser variability of mature skin. The variation in the measured parameters suggests that variation is dominated by tissue heterogeneity. Most users measured within one standard deviation of the group mean. Notably, corrected intrinsic fluorescence showed low intra- and interuser variability. These results strongly suggest that variability is mostly caused by tissue heterogeneity and is not user induced.

  15. Computational cell quantification in the human brain tissues based on hard x-ray phase-contrast tomograms

    NASA Astrophysics Data System (ADS)

    Hieber, Simone E.; Bikis, Christos; Khimchenko, Anna; Schulz, Georg; Deyhle, Hans; Thalmann, Peter; Chicherova, Natalia; Rack, Alexander; Zdora, Marie-Christine; Zanette, Irene; Schweighauser, Gabriel; Hench, Jürgen; Müller, Bert

    2016-10-01

    Cell visualization and counting plays a crucial role in biological and medical research including the study of neurodegenerative diseases. The neuronal cell loss is typically determined to measure the extent of the disease. Its characterization is challenging because the cell density and size already differs by more than three orders of magnitude in a healthy cerebellum. Cell visualization is commonly performed by histology and fluorescence microscopy. These techniques are limited to resolve complex microstructures in the third dimension. Phase- contrast tomography has been proven to provide sufficient contrast in the three-dimensional imaging of soft tissue down to the cell level and, therefore, offers the basis for the three-dimensional segmentation. Within this context, a human cerebellum sample was embedded in paraffin and measured in local phase-contrast mode at the beamline ID19 (ESRF, Grenoble, France) and the Diamond Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK). After the application of Frangi-based filtering the data showed sufficient contrast to automatically identify the Purkinje cells and to quantify their density to 177 cells per mm3 within the volume of interest. Moreover, brain layers were segmented in a region of interest based on edge detection. Subsequently performed histological analysis validated the presence of the cells, which required a mapping from the two- dimensional histological slices to the three-dimensional tomogram. The methodology can also be applied to further tissue types and shows potential for the computational tissue analysis in health and disease.

  16. Surfactant-aided precipitation/on-pellet-digestion (SOD) procedure provides robust and rapid sample preparation for reproducible, accurate and sensitive LC/MS quantification of therapeutic protein in plasma and tissues.

    PubMed

    An, Bo; Zhang, Ming; Johnson, Robert W; Qu, Jun

    2015-04-07

    For targeted protein quantification by liquid chromatography mass spectrometry (LC/MS), an optimal approach for efficient, robust and hi-throughput sample preparation is critical, but often remains elusive. Here we describe a straightforward surfactant-aided-precipitation/on-pellet-digestion (SOD) strategy that provides effective sample cleanup and enables high and constant peptide yields in various matrices, allowing reproducible, accurate and sensitive protein quantification. This strategy was developed using quantification of monocolnocal antibody in tissues and plasma as the model system. Surfactant treatment before precipitation substantially increased peptide recovery and reproducibility from plasma/tissue, likely because surfactant permits extensive denaturation/reduction/alkylation of proteins and inactivation of endogenous protease inhibitors, and facilitates removal of matrix components. The subsequent precipitation procedure effectively eliminates the surfactant and nonprotein matrix components, and the thorough denaturation by both surfactant and precipitation enabled very rapid on-pellet-digestion (45 min at 37 °C) with high peptide recovery. The performance of SOD was systematically compared against in-solution-digestion, in-gel-digestion and filter-aided-sample-preparation (FASP) in plasma/tissues, and then examined in a full pharmacokinetic study in rats. SOD achieved the best peptide recovery (∼21.0-700% higher than the other three methods across various matrices), reproducibility (3.75-10.9%) and sensitivity (28-30 ng/g across plasma and tissue matrices), and its performance was independent of matrix types. Finally, in validation and pharmacokinetic studies in rats, SOD outperformed other methods and provided highly accurate and precise quantification in all plasma samples without using stable isotope labeled (SIL)-protein internal standard (I.S.). In summary, the SOD method has proven to be highly robust, efficient and rapid, making it readily

  17. Validation of a fast method for quantification of intra-abdominal and subcutaneous adipose tissue for large-scale human studies.

    PubMed

    Borga, Magnus; Thomas, E Louise; Romu, Thobias; Rosander, Johannes; Fitzpatrick, Julie; Dahlqvist Leinhard, Olof; Bell, Jimmy D

    2015-12-01

    Central obesity is the hallmark of a number of non-inheritable disorders. The advent of imaging techniques such as MRI has allowed for a fast and accurate assessment of body fat content and distribution. However, image analysis continues to be one of the major obstacles to the use of MRI in large-scale studies. In this study we assess the validity of the recently proposed fat-muscle quantitation system (AMRA(TM) Profiler) for the quantification of intra-abdominal adipose tissue (IAAT) and abdominal subcutaneous adipose tissue (ASAT) from abdominal MR images. Abdominal MR images were acquired from 23 volunteers with a broad range of BMIs and analysed using sliceOmatic, the current gold-standard, and the AMRA(TM) Profiler based on a non-rigid image registration of a library of segmented atlases. The results show that there was a highly significant correlation between the fat volumes generated by the two analysis methods, (Pearson correlation r = 0.97, p < 0.001), with the AMRA(TM) Profiler analysis being significantly faster (~3 min) than the conventional sliceOmatic approach (~40 min). There was also excellent agreement between the methods for the quantification of IAAT (AMRA 4.73 ± 1.99 versus sliceOmatic 4.73 ± 1.75 l, p = 0.97). For the AMRA(TM) Profiler analysis, the intra-observer coefficient of variation was 1.6% for IAAT and 1.1% for ASAT, the inter-observer coefficient of variation was 1.4% for IAAT and 1.2% for ASAT, the intra-observer correlation was 0.998 for IAAT and 0.999 for ASAT, and the inter-observer correlation was 0.999 for both IAAT and ASAT. These results indicate that precise and accurate measures of body fat content and distribution can be obtained in a fast and reliable form by the AMRA(TM) Profiler, opening up the possibility of large-scale human phenotypic studies.

  18. Quantification of particles of lethal mercury in mouse viscera: high-resolution study of mercury in cells and tissues.

    PubMed

    Cunha, Elisabete M; Cherdwongcharoensuk, D; Aguas, Artur P

    2003-07-01

    To investigate the early visceral distribution of mercury (Hg), we have intraperitoneally injected a lethal dose of HgCl2 that killed BALB/c mice within 2-4 min. Scanning electron microscopy coupled with X-ray microanalysis (SEM-XRM) was used to detect and quantify Hg in situ in different organs. The highest density of Hg was seen in the liver (60.9+/-24.9 Hg particles per mm2 of tissue); this density was three and six times higher than those of renal or splenic Hg, respectively. Hg was scarce in the lungs and absent in the brain. Considering the relative weights of mouse viscera, our quantitative data show that the liver captured 89% of the visceral Hg; the kidneys captured 8.5% and the spleen just 1.7%. SEM-XRM revealed that most of the visceral Hg was associated with resident macrophages, a few Hg dots being detected on the surface of erythrocytes. We conclude that: (i) most intraperitoneally injected Hg was captured by liver Kupffer cells within minutes of injection; (ii) a 10-fold lower density of Hg particles was observed in the kidneys, and a 50-fold lower deposition of Hg was found in the spleen; (iii) SEM-XRM is an adequate method to quantify microparticles of Hg in tissues and cells.

  19. Quantification of telomere features in tumor tissue sections by an automated 3D imaging-based workflow.

    PubMed

    Gunkel, Manuel; Chung, Inn; Wörz, Stefan; Deeg, Katharina I; Simon, Ronald; Sauter, Guido; Jones, David T W; Korshunov, Andrey; Rohr, Karl; Erfle, Holger; Rippe, Karsten

    2017-02-01

    The microscopic analysis of telomere features provides a wealth of information on the mechanism by which tumor cells maintain their unlimited proliferative potential. Accordingly, the analysis of telomeres in tissue sections of patient tumor samples can be exploited to obtain diagnostic information and to define tumor subgroups. In many instances, however, analysis of the image data is conducted by manual inspection of 2D images at relatively low resolution for only a small part of the sample. As the telomere feature signal distribution is frequently heterogeneous, this approach is prone to a biased selection of the information present in the image and lacks subcellular details. Here we address these issues by using an automated high-resolution imaging and analysis workflow that quantifies individual telomere features on tissue sections for a large number of cells. The approach is particularly suited to assess telomere heterogeneity and low abundant cellular subpopulations with distinct telomere characteristics in a reproducible manner. It comprises the integration of multi-color fluorescence in situ hybridization, immunofluorescence and DNA staining with targeted automated 3D fluorescence microscopy and image analysis. We apply our method to telomeres in glioblastoma and prostate cancer samples, and describe how the imaging data can be used to derive statistically reliable information on telomere length distribution or colocalization with PML nuclear bodies. We anticipate that relating this approach to clinical outcome data will prove to be valuable for pretherapeutic patient stratification.

  20. Quantification of endogenous retinoids.

    PubMed

    Kane, Maureen A; Napoli, Joseph L

    2010-01-01

    Numerous physiological processes require retinoids, including development, nervous system function, immune responsiveness, proliferation, differentiation, and all aspects of reproduction. Reliable retinoid quantification requires suitable handling and, in some cases, resolution of geometric isomers that have different biological activities. Here we describe procedures for reliable and accurate quantification of retinoids, including detailed descriptions for handling retinoids, preparing standard solutions, collecting samples and harvesting tissues, extracting samples, resolving isomers, and detecting with high sensitivity. Sample-specific strategies are provided for optimizing quantification. Approaches to evaluate assay performance also are provided. Retinoid assays described here for mice also are applicable to other organisms including zebrafish, rat, rabbit, and human and for cells in culture. Retinoid quantification, especially that of retinoic acid, should provide insight into many diseases, including Alzheimer's disease, type 2 diabetes, obesity, and cancer.

  1. Quantification of collagen fiber organization in biological tissues at cellular and molecular scales using second-harmonic generation imaging

    NASA Astrophysics Data System (ADS)

    Ambekar Ramachandra Rao, Raghu

    Collagen is the most abundant structural protein found in the human body, and is responsible for providing structure and function to tissues. Collagen molecules organize naturally into structures called fibers on the scale of the wavelength of light and lack inversion symmetry, thus allowing for the process of second harmonic generation (SHG) when exposed to intense incident light. We have developed two quantitative techniques: Fourier transform-second-harmonic generation (FT-SHG) imaging and generalized chi2 second-harmonic generation (chi2-SHG) imaging. In order to show that FT-SHG imaging can be used as a valuable diagnostic tool for real-world biological problems, we first investigate collagenase-induced injury in horse tendons. Clear differences in collagen fiber organization between normal and injured tendon are quantified. In particular, we observe that the regularly oriented organization of collagen fibers in normal tendons is disrupted in injured tendons leading to a more random organization. We also observe that FT-SHG microscopy is more sensitive in assessing tendon injury compared to the conventional polarized light microscopy. The second study includes quantifying collagen fibers in cortical bone using FT-SHG imaging and comparing it with scanning electron microscopy (SEM). Further, as an example study, we show how FT-SHG imaging could be used to quantify changes in bone structure as a function of age. Some initial work and future directions for extending FT-SHG to 3D are also discussed. The second technique, chi2-SHG imaging, takes advantage of the coherent nature of SHG and utilizes polarization to extract the second-order susceptibility (d elements) which provides information on molecular organization, i.e., it provides access to sub-diffractional changes "optically". We use chi2-SHG in combination with FT-SHG imaging to investigate a couple of biological problems. First, we quantify differences in collagen fiber organization between cornea and

  2. Quantification of β-Catenin Signaling Components in Colon Cancer Cell Lines, Tissue Sections, and Microdissected Tumor Cells using Reaction Monitoring Mass Spectrometry

    PubMed Central

    Chen, Yi; Gruidl, Mike; Remily-Wood, Elizabeth; Liu, Richard Z.; Eschrich, Steven; Lloyd, Mark; Nasir, Aejaz; Bui, Marilyn M.; Huang, Emina; Shibata, David; Yeatman, Timothy; Koomen, John M.

    2010-01-01

    Reaction monitoring mass spectrometry has emerged as a powerful tool for targeted detection and quantification of proteins in clinical samples. Here, we report the use of gel electrophoresis for protein fractionation and liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) screening for quantitative analysis of components from the Wnt/β-catenin signaling pathway, which contributes to colon tumor formation and progression. In silico tools are used to design LC-MRM screens for each target protein. Following successful peptide detection, stable isotope labeled peptides are synthesized and developed as internal standards. Then, the assays are implemented in colon cancer cell lines to achieve detection in minimal amounts of cells, compatible with direct translation to clinical specimens. Selected assays are compared with qualitative results from immunoblotting (Westerns) and translated to individual frozen colon tissue sections and laser capture microdissected tumor cells. This LC-MRM platform has been translated from in vitro models to clinical specimens, forming the basis for future experiments in patient assessment. PMID:20590165

  3. Quantification of Candesartan in Mouse Plasma by MALDI-TOFMS and in Tissue Sections by MALDI-Imaging Using the Stable-Isotope Dilution Technique

    PubMed Central

    Nakanishi, Toyofumi; Takai, Shinji; Jin, Denan; Takubo, Takayuki

    2013-01-01

    To determine the contents of candesartan in mouse plasma, and blood vessel and kidney sliced sections and also better understand its pharmacokinetics, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and MALDI-imaging mass spectrometry (IMS) with the selected reaction monitoring (SRM) mode using a labeled-internal standard. The results of fundamental examinations showed that the slope of the resulting curves of candesartan in the plasma from the equation was 0.91 and the y-intercept was 0.02. Both intra- and inter-day accuracies (n=10) and the precision of candesartan in the plasma by MALDI-TOFMS with the SRM mode were in the range of 3.4 to 17.3% and 93.2%, respectively. The detection limit of candesartan in spiked plasma was 0.2 nmol/L. IMS analysis enabled us to clarify distinct spacial time-distribution images in sliced mouse blood vessel and kidney sections although it still needed to improve a protocol of quantification. Typical pharmacokinetic patterns of candesartan were obtained in the plasma and sliced kidney sections, but those in the blood vessel sections gradually increased 24 h after administration. MALDI-TOFMS and IMS with the SRM mode are powerful tools to identify the spacial distribution and traceability of candesartan in sliced blood vessel and tissue sections as well as in the plasma. PMID:24860711

  4. Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

    USGS Publications Warehouse

    Chase, D.M.; Elliott, D.G.; Pascho, R.J.

    2006-01-01

    Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

  5. Heavy Metal Quantification in Renal Tissue of Patients in the State of Yucatan and Its Association with Urolithiasis

    PubMed Central

    May-Ix, Luis A.; Rosado-Rubio, J. Gabriel; Medina-Escobedo, Martha; Castellanos-Ruelas, Arturo F.; Chel-Guerrero, Luis A.; Betancur-Ancona, David A.

    2012-01-01

    A possible cause associated with urinary lithiasis (UL) is the bioaccumulation of heavy metals in the kidney. The aim of this study was to evaluate the content of Cu, Pb, and Cd in kidney tissues removed from patients with nephrological problems and associate it with UL. Samples of 50 kidney sections from patients were analyzed. Results were statistically analyzed using a fixed effects model including the overall mean, the effect of the health status of patients (with or without UL), gender (male and female), the interaction between both factors and the random error (NID  (0, σ2)). Cu level was 8.8 ± 4.4 mg/kg (mean ± DS) and 25.5% of samples had levels above normal. Lead content in 97.9% of the samples (3.6 ± 1.5 mg/kg) was above normal. All results of Cd (13.2 ± 16.6 mg/kg) were below the maximum permissible limits. There was no difference in the amount of heavy metals on patients with or without UL (P > 0.05) nor depending on the gender (P > 0.05). It was concluded that there is no apparent relationship between a very elevated level of Cu or Pb in the kidney on the development of UL. PMID:23762635

  6. A new method of infrared thermography for quantification of brown adipose tissue activation in healthy adults (TACTICAL): a randomized trial.

    PubMed

    Ang, Qi Yan; Goh, Hui Jen; Cao, Yanpeng; Li, Yiqun; Chan, Siew-Pang; Swain, Judith L; Henry, Christiani Jeyakumar; Leow, Melvin Khee-Shing

    2017-05-01

    The ability to alter the amount and activity of brown adipose tissue (BAT) in human adults is a potential strategy to manage obesity and related metabolic disorders associated with food, drug, and environmental stimuli with BAT activating/recruiting capacity. Infrared thermography (IRT) provides a non-invasive and inexpensive alternative to the current methods (e.g. (18)F-FDG PET) used to assess BAT. We have quantified BAT activation in the cervical-supraclavicular (C-SCV) region using IRT video imaging and a novel image computational algorithm by studying C-SCV heat production in healthy young men after cold stimulation and the ingestion of capsinoids in a prospective double-blind placebo-controlled randomized trial. Subjects were divided into low-BAT and high-BAT groups based on changes in IR emissions in the C-SCV region induced by cold. The high-BAT group showed significant increases in energy expenditure, fat oxidation, and heat output in the C-SCV region post-capsinoid ingestion compared to post-placebo ingestion, but the low-BAT group did not. Based on these results, we conclude that IRT is a promising tool for quantifying BAT activity.

  7. Virtual touch tissue imaging and quantification (VTIQ) in the evaluation of thyroid nodules: the associated factors leading to misdiagnosis

    PubMed Central

    Sun, Cheng-Yu; Lei, Kai-Rong; Liu, Bo-Ji; Bo, Xiao-Wan; Li, Xiao-Long; He, Ya-Ping; Wang, Dan; Ren, Wei-Wei; Zhao, Chong-Ke; Xu, Hui-Xiong

    2017-01-01

    To evaluate the associated factors leading to misdiagnosis with VTIQ for differentiation between benign from malignant thyroid nodules (TNs). The study included 238 benign TNs and 150 malignant TNs. Conventional ultrasound (US) features and VTIQ parameters were obtained and compared with the reference standard of histopathological and/or cytological results. Binary logistic regression analysis was performed to select independent variables leading to misdiagnosis. The maximum shear wave speed (SWS) (SWS-max), mean SWS (SWS-mean), SWS-ratio and standard deviation of SWS (SWS-SD) were significantly higher for malignant TNs compared with benign TNs (all P < 0.001). SWS-mean achieved the highest diagnostic performance with a cut-off value of 3.15 m/s. False positive rate was 13.4% (32/238) while false negative rate was 35.3% (53/150). Intranodular calcification (OR: 1.715) was significantly associated with false positive VTIQ findings, while nodule size (OR: 0.936) and echotexture of the thyroid gland (OR: 0.033) were negatively associated with them. Nodule depth (OR: 0.881) and TI-RADS category (OR: 0.563) were negatively associated with false negative VTIQ findings. These US characteristic of TNs should be taken into consideration when interpreting the results of VTIQ examinations. PMID:28157195

  8. Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization.

    PubMed

    Sudhakar Reddy, Palakolanu; Srinivas Reddy, Dumbala; Sivasakthi, Kaliamoorthy; Bhatnagar-Mathur, Pooja; Vadez, Vincent; Sharma, Kiran K

    2016-01-01

    Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.

  9. Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization

    PubMed Central

    Sudhakar Reddy, Palakolanu; Srinivas Reddy, Dumbala; Sivasakthi, Kaliamoorthy; Bhatnagar-Mathur, Pooja; Vadez, Vincent; Sharma, Kiran K.

    2016-01-01

    Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species. PMID:27200008

  10. Certification of methylmercury in cod fish tissue certified reference material by species-specific isotope dilution mass spectrometric analysis.

    PubMed

    Inagaki, Kazumi; Kuroiwa, Takayoshi; Narukawa, Tomohiro; Yarita, Takashi; Takatsu, Akiko; Okamoto, Kensaku; Chiba, Koichi

    2008-07-01

    A new cod fish tissue certified reference material, NMIJ CRM 7402-a, for methylmercury analysis was certified by the National Metrological Institute of Japan in the National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). Cod fish was collected from the sea close to Japan. The cod muscle was powdered by freeze-pulverization and was placed into 600 glass bottles (10 g each), which were sterilized with gamma-ray irradiation. The certification was carried out using species-specific isotope dilution gas chromatography inductively coupled plasma mass spectrometry (SSID-GC-ICPMS), where (202)Hg-enriched methylmercury (MeHg) was used as the spike compound. In order to avoid any possible analytical biases caused by nonquantitative extraction, degradation and/or formation of MeHg in sample preparations, two different extraction methods (KOH/methanol and HCl/methanol extractions) were performed, and one of these extraction methods utilized two different derivatization methods (ethylation and phenylation). A double ID method was adopted to minimize the uncertainty arising from the analyses. In order to ensure not only the reliability of the analytical results but also traceability to SI units, the standard solution of MeHg used for the reverse-ID was prepared from high-purity MeHg chloride and was carefully assayed as follows: the total mercury was determined by ID-ICPMS following aqua regia digestion, and the ratio of Hg as MeHg to the total Hg content was estimated by GC-ICPMS. The certified value given for MeHg is 0.58 +/- 0.02 mg kg(-1) as Hg.

  11. Reference gene selection for quantitative gene expression studies during biological invasions: A test on multiple genes and tissues in a model ascidian Ciona savignyi.

    PubMed

    Huang, Xuena; Gao, Yangchun; Jiang, Bei; Zhou, Zunchun; Zhan, Aibin

    2016-01-15

    As invasive species have successfully colonized a wide range of dramatically different local environments, they offer a good opportunity to study interactions between species and rapidly changing environments. Gene expression represents one of the primary and crucial mechanisms for rapid adaptation to local environments. Here, we aim to select reference genes for quantitative gene expression analysis based on quantitative Real-Time PCR (qRT-PCR) for a model invasive ascidian, Ciona savignyi. We analyzed the stability of ten candidate reference genes in three tissues (siphon, pharynx and intestine) under two key environmental stresses (temperature and salinity) in the marine realm based on three programs (geNorm, NormFinder and delta Ct method). Our results demonstrated only minor difference for stability rankings among the three methods. The use of different single reference gene might influence the data interpretation, while multiple reference genes could minimize possible errors. Therefore, reference gene combinations were recommended for different tissues - the optimal reference gene combination for siphon was RPS15 and RPL17 under temperature stress, and RPL17, UBQ and TubA under salinity treatment; for pharynx, TubB, TubA and RPL17 were the most stable genes under temperature stress, while TubB, TubA and UBQ were the best under salinity stress; for intestine, UBQ, RPS15 and RPL17 were the most reliable reference genes under both treatments. Our results suggest that the necessity of selection and test of reference genes for different tissues under varying environmental stresses. The results obtained here are expected to reveal mechanisms of gene expression-mediated invasion success using C. savignyi as a model species.

  12. Evaluation of candidate reference genes for RT-qPCR studies in three metabolism related tissues of mice after caloric restriction

    PubMed Central

    Gong, Huan; Sun, Liang; Chen, Beidong; Han, Yiwen; Pang, Jing; Wu, Wei; Qi, Ruomei; Zhang, Tie-mei

    2016-01-01

    Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) is a routine method for gene expression analysis, and reliable results depend on proper normalization by stable reference genes. Caloric restriction (CR) is a robust lifestyle intervention to slow aging and delay onset of age-associated diseases via inducing global changes in gene expression. Reliable normalization of RT-qPCR data becomes crucial in CR studies. In this study, the expression stability of 12 candidate reference genes were evaluated in inguinal white adipose tissue (iWAT), skeletal muscle (Sk.M) and liver of CR mice by using three algorithms, geNorm, NormFinder, and Bestkeeper. Our results showed β2m, Ppia and Hmbs as the most stable genes in iWAT, Sk.M and liver, respectively. Moreover, two reference genes were sufficient to normalize RT-qPCR data in each tissue and the suitable pair of reference genes was β2m-Hprt in iWAT, Ppia-Gusb in Sk.M and Hmbs-β2m in liver. By contrast, the least stable gene in iWAT or Sk.M was Gapdh, and in liver was Pgk1. Furthermore, the expression of Leptin and Ppar-γ were profiled in these tissues to validate the selected reference genes. Our data provided a basis for gene expression analysis in future CR studies. PMID:27922100

  13. Precise simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples following their successive i.p. administration.

    PubMed

    Nakhla, David S; Hussein, Lobna A; Magdy, N; Abdallah, Inas A; Hassan, Hazem E

    2017-03-24

    A sensitive high-performance liquid chromatography (HPLC) assay with dual UV detection has been developed and validated for the simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples. Liquid-liquid extraction using hexanes was applied for samples extraction with Levo-Tetrahydropalmatine (L-THP) as the internal standard. Chromatographic separation of the analytes was achieved on a reversed-phase Waters Symmetry(®) C18 column (150mm×4.6mm, 5μm). A gradient elution was employed with a mobile phase consisting of 5mM potassium phosphate containing 0.1% triethylamine (pH=6.5) (A) and acetonitrile (B) with a flow rate of 1mL/min. UV detection was employed at 215nm and 235nm for the determination of methadone and cocaine, respectively. The calibration curves were linear over the range of 0.05-10μg/mL for both methadone and cocaine. The assay was validated according to FDA guidelines for bioanalytical method validation and results were satisfactory and met FDA criteria. Inter-day accuracy values of serum and brain samples ranged from 96.97 to 105.59% while intra-day accuracy values ranged from 91.49 to 111.92%. Stability assays showed that both methadone and cocaine were stable during sample storage, preparation, and analytical procedures. The method was successfully used to analyze biological samples obtained from a drug- drug interaction pharmacokinetics (PK) study conducted in rats to investigate the effect of methadone on cocaine PK. Our method not only can be used for bioanalysis of samples obtained from rats but also can potentially be applied to human biological serum samples to monitor compliance to methadone maintenance therapy (MMT) and to detect possible cocaine-methadone co-abuse.

  14. Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions.

    PubMed

    Svingen, Terje; Letting, Heidi; Hadrup, Niels; Hass, Ulla; Vinggaard, Anne Marie

    2015-01-01

    In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or 'housekeeping') gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a, Rps18, Rps29, Sdha, Tbp and Ubc) across several juvenile and adult rat tissues (liver, adrenal, prostate, fat pad, testis and ovaries), both under normal conditions and following exposure to various chemicals during development. Employing NormFinder and BestKeeper softwares, we found Hprt and Sdha to be amongst the most stable genes across normal and manipulated tissues, with several others also being suitable for most tissues. Tbp and B2m displayed highest variability in transcript levels between tissues and developmental stages. It was also observed that the reference genes were most unstable in liver and testis following toxicological exposure. For future studies, we propose the use of more than one verified reference gene and the continuous monitoring of their suitability under various experimental conditions, including toxicological studies, based on changes in threshold (Ct) values from cDNA samples having been reverse-transcribed from a constant input concentration of RNA.

  15. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    PubMed

    Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  16. Quantification of brain lipids by FTIR spectroscopy and partial least squares regression

    NASA Astrophysics Data System (ADS)

    Dreissig, Isabell; Machill, Susanne; Salzer, Reiner; Krafft, Christoph

    2009-01-01

    Brain tissue is characterized by high lipid content. Its content decreases and the lipid composition changes during transformation from normal brain tissue to tumors. Therefore, the analysis of brain lipids might complement the existing diagnostic tools to determine the tumor type and tumor grade. Objective of this work is to extract lipids from gray matter and white matter of porcine brain tissue, record infrared (IR) spectra of these extracts and develop a quantification model for the main lipids based on partial least squares (PLS) regression. IR spectra of the pure lipids cholesterol, cholesterol ester, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, galactocerebroside and sulfatide were used as references. Two lipid mixtures were prepared for training and validation of the quantification model. The composition of lipid extracts that were predicted by the PLS regression of IR spectra was compared with lipid quantification by thin layer chromatography.

  17. Quantification of five compounds with heterogeneous physicochemical properties (morphine, 6-monoacetylmorphine, cyamemazine, meprobamate and caffeine) in 11 fluids and tissues, using automated solid-phase extraction and gas chromatography-tandem mass spectrometry.

    PubMed

    Bévalot, Fabien; Bottinelli, Charline; Cartiser, Nathalie; Fanton, Laurent; Guitton, Jérôme

    2014-06-01

    An automated solid-phase extraction (SPE) protocol followed by gas chromatography coupled with tandem mass spectrometry was developed for quantification of caffeine, cyamemazine, meprobamate, morphine and 6-monoacetylmorphine (6-MAM) in 11 biological matrices [blood, urine, bile, vitreous humor, liver, kidney, lung and skeletal muscle, brain, adipose tissue and bone marrow (BM)]. The assay was validated for linearity, within- and between-day precision and accuracy, limits of quantification, selectivity, extraction recovery (ER), sample dilution and autosampler stability on BM. For the other matrices, partial validation was performed (limits of quantification, linearity, within-day precision, accuracy, selectivity and ER). The lower limits of quantification were 12.5 ng/mL(ng/g) for 6-MAM, morphine and cyamemazine, 100 ng/mL(ng/g) for meprobamate and 50 ng/mL(ng/g) for caffeine. Analysis of real-case samples demonstrated the performance of the assay in forensic toxicology to investigate challenging cases in which, for example, blood is not available or in which analysis in alternative matrices could be relevant. The SPE protocol was also assessed as an extraction procedure that could target other relevant analytes of interest. The extraction procedure was applied to 12 molecules of forensic interest with various physicochemical properties (alimemazine, alprazolam, amitriptyline, citalopram, cocaine, diazepam, levomepromazine, nordazepam, tramadol, venlafaxine, pentobarbital and phenobarbital). All drugs were able to be detected at therapeutic concentrations in blood and in the alternate matrices.

  18. Dynamic modeling of breast tissue with application of model reference adaptive system identification technique based on clinical robot-assisted palpation.

    PubMed

    Keshavarz, M; Mojra, A

    2015-11-01

    Accurate identification of breast tissue's dynamic behavior in physical examination is critical to successful diagnosis and treatment. In this study a model reference adaptive system identification (MRAS) algorithm is utilized to estimate the dynamic behavior of breast tissue from mechanical stress-strain datasets. A robot-assisted device (Robo-Tac-BMI) is going to mimic physical palpation on a 45 year old woman having a benign mass in the left breast. Stress-strain datasets will be collected over 14 regions of both breasts in a specific period of time. Then, a 2nd order linear model is adapted to the experimental datasets. It was confirmed that a unique dynamic model with maximum error about 0.89% is descriptive of the breast tissue behavior meanwhile mass detection may be achieved by 56.1% difference from the normal tissue.

  19. Quantification of drugs in plasma without primary reference standards by liquid chromatography-chemiluminescence nitrogen detection: application to tramadol metabolite ratios.

    PubMed

    Ojanperä, Suvi; Rasanen, Ilpo; Sistonen, Johanna; Pelander, Anna; Vuori, Erkki; Ojanperä, Ilkka

    2007-08-01

    Lack of availability of reference standards for drug metabolites, newly released drugs, and illicit drugs hinders the analysis of these substances in biologic samples. To counter this problem, an approach is presented here for quantitative drug analysis in plasma without primary reference standards by liquid chromatography-chemiluminescence nitrogen detection (LC-CLND). To demonstrate the feasibility of the method, metabolic ratios of the opioid drug tramadol were determined in the setting of a pharmacogenetic study. Four volunteers were given a single 100-mg oral dose of tramadol, and a blood sample was collected from each subject 1 hour later. Tramadol, O-desmethyltramadol, and nortramadol were determined in plasma by LC-CLND without reference standards and by a gas chromatography-mass spectrometry reference method. In contrast to previous CLND studies lacking an extraction step, a liquid-liquid extraction system was created for 5-mL plasma samples using n-butyl chloride-isopropyl alcohol (98 + 2) at pH 10. Extraction recovery estimation was based on model compounds chosen according to their similar physicochemical characteristics (retention time, pKa, logD). Instrument calibration was performed with a single secondary standard (caffeine) using the equimolar response of the detector to nitrogen. The mean differences between the results of the LC-CLND and gas chromatography-mass spectrometry methods for tramadol, O-desmethyltramadol, and nortramadol were 8%, 32%, and 19%, respectively. The sensitivity of LC-CLND was sufficient for therapeutic concentrations of tramadol and metabolites. A good correlation was obtained between genotype, expressed by the number of functional genes, and the plasma metabolite ratios. This experiment suggests that a recovery-corrected LC-CLND analysis produces sufficiently accurate results to be useful in a clinical context, particularly in instances in which reference standards are not readily accessible.

  20. Development and reproducibility of a 3D stereophotogrammetric reference frame for facial soft tissue growth of babies and young children with and without orofacial clefts.

    PubMed

    Brons, S; van Beusichem, M E; Maal, T J J; Plooij, J M; Bronkhorst, E M; Bergé, S J; Kuijpers-Jagtman, A M

    2013-01-01

    The aim of this study was to develop a reference frame for three dimensional (3D) facial soft tissue growth analysis in children and to determine its reproducibility. Two observers twice placed the reference frame on 39 3D-stereophotogrammetry facial images of children with orofacial clefts and control children. The observers' performances were analyzed by calculating mean distance, distance variability, and P95 between the same facial surfaces at two different time points. Correlations between observers were analyzed with Pearson's correlation coefficient. The influence of presence of a cleft, absence of one ear in the photograph, and age on the reproducibility of the reference frame was checked using Student's t test. Results of intraobserver comparisons showed a mean distance of <0.40 mm, distance variability of <0.51 mm, and P95 of <0.80 mm. For interobserver reliability, the mean distance was <0.52 mm, distance variability was <0.53 mm, and P95 was <1.10 mm. Presence of a cleft, age, and absence of one ear on the 3D photograph did not have a significant influence on the reproducibility of placing the reference frame. The children's reference frame is a reproducible method to superimpose on 3D soft tissue stereophotogrammetry photographs of growing individuals with and without orofacial clefts.

  1. Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation

    PubMed Central

    Masè, Michela; Grasso, Margherita; Avogaro, Laura; D’Amato, Elvira; Tessarolo, Francesco; Graffigna, Angelo; Denti, Michela Alessandra; Ravelli, Flavia

    2017-01-01

    MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-Cq, GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions. PMID:28117343

  2. Structure-function relationships in radiation-induced cell and tissue lesions: special references to the contributions of scanning electron microscopy and hematopoietic tissue responses

    SciTech Connect

    Seed, T.M.

    1987-03-01

    Contributions of scanning electron microscopy to the field of radiation biology are briefly reviewed and presented in terms of an overall goal to identify and characterize the structural features of radiation-induced lesions in vital cell and tissue targets. In the context of lesion production, the major radiation-elicited response sequences, the types and nature of measured end points, and governing temporal and radiobiological parameters are discussed and illustrated by using results derived from both in vitro cell systems and in vivo studies that measured tissue responses from various organ systems (respiratory, digestive, circulatory, and central nervous systems). Work in our laboratory on the nature of early and late hematopathologic tissue responses (aplastic anemia and myeloid leukemia) induced by protracted radiation exposure and the bridging effect of repair processes relative to the expression of these pathologies is highlighted.

  3. Quantification of acylglycines in human urine by HPLC electrospray ionization-tandem mass spectrometry and the establishment of pediatric reference interval in local Chinese.

    PubMed

    Fong, Bonnie Mei-Wah; Tam, Sidney; Leung, Kelvin Sze-Yin

    2012-01-15

    Urinary organic acids, plasma amino acids and acylcarnitine profile analyses are the main tools used to diagnose inborn errors of metabolisms (IEMs). However, without metabolic decompensation, these parameters are often not helpful. On the other hand, in cases of IEM, acylglycines are consistently raised even when patients appear to be in remission. This study aims to set-up a simple liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of urine acylglycines, complementary to organic acid and acylcarnitine profiles, for the diagnosis of IEM. In addition, local reference intervals for various acylglycines are established by using this method. Acylglycines were isolated by solid-phase extraction, derivatized with n-butanol, separated by HPLC, and detected by ESI-MS/MS. Acylglycines were quantified with deuterated internal standards. Mean recoveries of acylglycines ranged from 90.2 to 109.3%. Within- and between-run imprecisions for all acylglycines have CVs less than 10%. Linear regression coefficients were greater than 0.99. Reference intervals were established according to CLSI guidelines by analyzing 204 samples from apparently healthy individuals less than 18 years of age. The distributions of AG in the "normal" urine were skewed towards the right. After log transformation, all the results were normally distributed. Partitioning into age group reference intervals was not indicated, according to the Harris and Boyd approach. In this context, a single reference interval for each acylglycine could be used. This method of urine acylglycines analysis is a powerful diagnostic tool, complementary to urine organic acids and plasma acylcarnitine profiling, for detecting certain inborn errors of metabolism.

  4. SNP calling from RNA-seq data without a reference genome: identification, quantification, differential analysis and impact on the protein sequence

    PubMed Central

    Lopez-Maestre, Hélène; Brinza, Lilia; Marchet, Camille; Kielbassa, Janice; Bastien, Sylvère; Boutigny, Mathilde; Monnin, David; Filali, Adil El; Carareto, Claudia Marcia; Vieira, Cristina; Picard, Franck; Kremer, Natacha; Vavre, Fabrice; Sagot, Marie-France; Lacroix, Vincent

    2016-01-01

    SNPs (Single Nucleotide Polymorphisms) are genetic markers whose precise identification is a prerequisite for association studies. Methods to identify them are currently well developed for model species, but rely on the availability of a (good) reference genome, and therefore cannot be applied to non-model species. They are also mostly tailored for whole genome (re-)sequencing experiments, whereas in many cases, transcriptome sequencing can be used as a cheaper alternative which already enables to identify SNPs located in transcribed regions. In this paper, we propose a method that identifies, quantifies and annotates SNPs without any reference genome, using RNA-seq data only. Individuals can be pooled prior to sequencing, if not enough material is available from one individual. Using pooled human RNA-seq data, we clarify the precision and recall of our method and discuss them with respect to other methods which use a reference genome or an assembled transcriptome. We then validate experimentally the predictions of our method using RNA-seq data from two non-model species. The method can be used for any species to annotate SNPs and predict their impact on the protein sequence. We further enable to test for the association of the identified SNPs with a phenotype of interest. PMID:27458203

  5. SNP calling from RNA-seq data without a reference genome: identification, quantification, differential analysis and impact on the protein sequence.

    PubMed

    Lopez-Maestre, Hélène; Brinza, Lilia; Marchet, Camille; Kielbassa, Janice; Bastien, Sylvère; Boutigny, Mathilde; Monnin, David; Filali, Adil El; Carareto, Claudia Marcia; Vieira, Cristina; Picard, Franck; Kremer, Natacha; Vavre, Fabrice; Sagot, Marie-France; Lacroix, Vincent

    2016-11-02

    SNPs (Single Nucleotide Polymorphisms) are genetic markers whose precise identification is a prerequisite for association studies. Methods to identify them are currently well developed for model species, but rely on the availability of a (good) reference genome, and therefore cannot be applied to non-model species. They are also mostly tailored for whole genome (re-)sequencing experiments, whereas in many cases, transcriptome sequencing can be used as a cheaper alternative which already enables to identify SNPs located in transcribed regions. In this paper, we propose a method that identifies, quantifies and annotates SNPs without any reference genome, using RNA-seq data only. Individuals can be pooled prior to sequencing, if not enough material is available from one individual. Using pooled human RNA-seq data, we clarify the precision and recall of our method and discuss them with respect to other methods which use a reference genome or an assembled transcriptome. We then validate experimentally the predictions of our method using RNA-seq data from two non-model species. The method can be used for any species to annotate SNPs and predict their impact on the protein sequence. We further enable to test for the association of the identified SNPs with a phenotype of interest.

  6. The Circulatory and Metabolic Responses to Hypoxia in Humans – With Special Reference to Adipose Tissue Physiology and Obesity

    PubMed Central

    Heinonen, Ilkka H. A.; Boushel, Robert; Kalliokoski, Kari K.

    2016-01-01

    Adipose tissue metabolism and circulation play an important role in human health. It is well-known that adipose tissue mass is increased in response to excess caloric intake leading to obesity and further to local hypoxia and inflammatory signaling. Acute exercise increases blood supply to adipose tissue and mobilization of fat stores for energy. However, acute exercise during systemic hypoxia reduces subcutaneous blood flow in healthy young subjects, but the response in overweight or obese subjects remains to be investigated. Emerging evidence also indicates that exercise training during hypoxic exposure may provide additive benefits with respect to many traditional cardiovascular risk factors as compared to exercise performed in normoxia, but unfavorable effects of hypoxia have also been documented. These topics will be covered in this brief review dealing with hypoxia and adipose tissue physiology. PMID:27621722

  7. Selection of reference genes as internal controls for gene expression in tissues of red abalone Haliotis rufescens (Mollusca, Vetigastropoda; Swainson, 1822).

    PubMed

    López-Landavery, Edgar A; Portillo-López, Amelia; Gallardo-Escárate, Cristian; Del Río-Portilla, Miguel A

    2014-10-10

    The red abalone Haliotis rufescens is one of the most important species for aquaculture in Baja California, México, and despite this, few gene expression studies have been done in tissues such as gill, head and gonad. For this purpose, reverse transcription and quantitative real time PCR (RT-qPCR) is a powerful tool for gene expression evaluation. For a reliable analysis, however, it is necessary to select and validate housekeeping genes that allow proper transcription quantification. Stability of nine housekeeping genes (ACTB, BGLU, TUBB, CY, GAPDH, HPRTI, RPL5, SDHA and UBC) was evaluated in different tissues of red abalone (gill, head and gonad/digestive gland). Four-fold serial dilutions of cDNA (from 25 ngμL(-1) to 0.39 ngμL(-1)) were used to prepare the standard curve, and it showed gene efficiencies between 0.95 and 0.99, with R(2)=0.99. geNorm and NormFinder analysis showed that RPL5 and CY were the most stable genes considering all tissues, whereas in gill HPRTI and BGLU were most stable. In gonad/digestive gland, RPL5 and TUBB were the most stable genes with geNorm, while SDHA and HPRTI were the best using NormFinder. Similarly, in head the best genes were RPL5 and UBC with geNorm, and GAPDH and CY with NormFinder. The technical variability analysis with RPL5 and abalone gonad/digestive gland tissue indicated a high repeatability with a variation coefficient within groups ≤ 0.56% and between groups ≤ 1.89%. These results will help us for further research in reproduction, thermoregulation and endocrinology in red abalone.

  8. Upper incisor to Soft Tissue Plane (UI-STP): a new reference for diagnosis and planning in dentofacial deformities.

    PubMed

    Hernandez-Alfaro, Federico

    2010-09-01

    Planning in orthognathic surgery has been and still is an open issue. We have evolved from 2D classical cephalometric hard-tissue planning to 2D soft tissue planning, and finally to 3D and hard and soft tissue evaluation. This, to our knowledge, is the first description of a new Soft Tissue Plane (STP) and its relationship with the anterior position of the upper incisor (UI). Profile photographs of 110 "attractive individuals" with lips at rest or smiling and with upper incisor shown were used. The photographs used were of 65 professional models from two international agencies and 45 individuals considered most attractive in the internet forums, which included catwalk models and actors. In 86 cases (78.18 %), the incisor was located in front of the STP (A). In 15 cases (13.63%), it was on the plane (N); and in the remaining 9 cases (8.18%), it was behind (P). Despite the limitations of this study and based on our series, we can conclude that the upper incisor is located at or in front of the Soft Tissue Plane (STP) in 91.81% of the attractive facial profiles studied. On the other hand, the relative position of the upper incisor to the soft tissue plane (UI-STP) could be a useful diagnostic and planning tool in orthodontic and surgical management of dentofacial deformities.

  9. Certification of NIST standard reference material 2389a, amino acids in 0.1 mol/L HCl--quantification by ID LC-MS/MS.

    PubMed

    Lowenthal, Mark S; Yen, James; Bunk, David M; Phinney, Karen W

    2010-05-01

    An isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) measurement procedure was developed to accurately quantify amino acid concentrations in National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2389a-amino acids in 0.1 mol/L hydrochloric acid. Seventeen amino acids were quantified using selected reaction monitoring on a triple quadrupole mass spectrometer. LC-MS/MS results were compared to gravimetric measurements from the preparation of SRM 2389a-a reference material developed at NIST and intended for use in intra-laboratory calibrations and quality control. Quantitative mass spectrometry results and gravimetric values were statistically combined into NIST-certified mass fraction values with associated uncertainty estimates. Coefficients of variation (CV) for the repeatability of the LC-MS/MS measurements among amino acids ranged from 0.33% to 2.7% with an average CV of 1.2%. Average relative expanded uncertainty of the certified values including Types A and B uncertainties was 3.5%. Mean accuracy of the LC-MS/MS measurements with gravimetric preparation values agreed to within |1.1|% for all amino acids. NIST SRM 2389a will be available for characterization of routine methods for amino acid analysis and serves as a standard for higher-order measurement traceability. This is the first time an ID LC-MS/MS methodology has been applied for quantifying amino acids in a NIST SRM material.

  10. Identification of reliable reference genes for quantitative gene expression studies in oral squamous cell carcinomas compared to adjacent normal tissues in the F344 rat model.

    PubMed

    Peng, Xinjian; McCormick, David L

    2016-08-01

    Oral squamous cell carcinomas (OSCCs) induced in F344 rats by 4-nitroquinoline-1-oxide (4-NQO) demonstrate considerable phenotypic similarity to human oral cancers and the model has been widely used for carcinogenesis and chemoprevention studies. Molecular characterization of this model needs reliable reference genes (RGs) to avoid false- positive and -negative results for proper interpretation of gene expression data between tumor and adjacent normal tissues. Microarray analysis of 11 pairs of OSCC and site-matched phenotypically normal oral tissues from 4-NQO-treated rats identified 10 stably expressed genes in OSCC compared to adjacent normal tissues (p>0.5, CV<15%) that could serve as potential RGs in this model. The commonly used 27 RGs in the rat were also analyzed based on microarray data and most of them were found unsuitable for RGs in this model. Traditional RGs such as ACTB and GAPDH were significantly altered in OSCC compared to adjacent normal tissues (p<0.01, n=11); however, the Hsp90ab1 was ranked as the best RG candidate and the combination of Hsp90ab1 and HPRT1 was identified by NormFinder to be a superior reference for gene normalization among the commonly used RGs. This result was also validated by RT-PCR based on the selected top RG candidate pool. These data suggest that there are no common RGs suitable for different models and RG(s) should be identified before gene expression analysis. We successfully identified Hsp90ab1 as a stable RG in 4-NQO-induced OSCC compared to adjacent normal tissues in F344 rats. The combination of two stably expressed genes may be a better option for gene normalization in tissue samples.

  11. Development of certified matrix-based reference material of genetically modified rice event TT51-1 for real-time PCR quantification.

    PubMed

    Jiang, Yu; Yang, Hui; Quan, Sheng; Liu, Yinan; Shen, Ping; Yang, Litao

    2015-09-01

    In 2009, the genetically modified (GM) rice event TT51-1 with an engineered insect resistance trait became the first GM rice event to be granted certification for safe production in China, and its derivative lines Bt 63 and Huahui No.1 are expected to be commercialized soon. The development of certified reference material (CRM) for TT51-1 is necessary to monitor and inspect the TT51-1 event and its derivates. In this work, we developed four matrix-based TT51-1 rice CRMs (TT51-1a, TT51-1b, TT51-1c, and TT51-1d) with different TT51-1 mass fraction ratios by blending seed powders of homozygous TT51-1 and its recipient cultivar Minghui 63. The between-bottle homogeneity and the within-bottle homogeneity were tested, and good results were obtained. The potential degradation during transportation and shelf life were evaluated, and demonstrated an expiration period of at least 36 months. The characterization values of the four TT51-1 CRMs based on the mass fraction ratio were 1000.000 ± 51.430 g/kg, 49.940 ± 4.620 g/kg, 9.990 ± 1.110 g/kg, and 4.990 ± 0.620 g/kg, respectively. The characterization values based on the copy number ratio were certified by digital PCR analysis as 97.442 ± 5.253 %, 4.851 ± 0.486 %, 1.042 ± 0.135 %, and 0.556 ± 0.073 %, respectively. These results suggested that the TT51-1 matrix-based CRMs developed are of high quality and can be used as potential calibrators for TT51-1 GM rice inspection and monitoring.

  12. Effect of addition of antibiotics and an antioxidant on the stability of tissue reference materials for domoic acid, the amnesic shellfish poison.

    PubMed

    McCarron, Pearse; Burrell, Stephen; Hess, Philipp

    2007-04-01

    Five separate reference materials (RMs) were prepared from a mussel (Mytilus edulis) tissue containing domoic acid (DA) from scallop hepatopancreas (Pecten maximus). Homogenates were separately spiked with antibiotics, an antioxidant, or a combination of both. Control materials did not contain any additives and were prepared from lightly cooked and autoclaved mussel tissues. Stability studies were run over a 148-day period at three different temperature conditions: -20 degrees C, +4 degrees C and +40 degrees C. DA contents in all materials were characterised by HPLC-UV. Homogeneities were demonstrated at the beginning of the study, with coefficients of variance of less than 4% (n = 9). DA was stable at -20 degrees C in all materials. The control materials showed significant degradation after two days at +40 degrees C, and after eight days at +4 degrees C. Each of the materials containing additives demonstrated better stability during the initial period of the study. In addition there was no significant degradation in any of the materials with additives stored at +4 degrees C over the duration of the study. The material containing a combination of the antibiotics and the antioxidant displayed the best stability of all the materials. There was no significant reduction in DA concentration at all temperature conditions after eight days, and after 32 days the decrease at +40 degrees C was still <20 %. Following this, a DA laboratory reference material (LRM) was prepared and, based on previous results, spiked with both the antioxidant and antibiotics. A short-term stability study on this material gave similar results to the corresponding material in the additives study. This study shows that combined use of the additives investigated in the preparation of a mussel tissue reference material for DA ensures analyte stability for a period of up to eight days at temperatures of up to +40 degrees C, a condition that is particularly important when shipping test materials

  13. Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions.

    PubMed

    Cassan-Wang, Hua; Soler, Marçal; Yu, Hong; Camargo, Eduardo Leal O; Carocha, Victor; Ladouce, Nathalie; Savelli, Bruno; Paiva, Jorge A P; Leplé, Jean-Charles; Grima-Pettenati, Jacqueline

    2012-12-01

    Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses.

  14. A new specific reference gene based on growth hormone gene (GH1) used for detection and relative quantification of Aquadvantage® GM salmon (Salmo salar L.) in food products.

    PubMed

    Ben Hafsa, Ahmed; Nabi, Nesrine; Zellama, Mohamed Salem; Said, Khaled; Chaouachi, Maher

    2016-01-01

    Genetic transformation of fish is mainly oriented towards the improvement of growth for the benefit of the aquaculture. Actually, Atlantic salmon (Salmo salar) is the species most transformed to achieve growth rates quite large compared to the wild. To anticipate the presence of contaminations with GM salmon in fish markets and the lack of labeling regulations with a mandatory threshold, the proper methods are needed to test the authenticity of the ingredients. A quantitative real-time polymerase chain reaction (QRT-PCR) method was used in this study. Ct values were obtained and validated using 15 processed food containing salmon. The relative and absolute limits of detection were 0.01% and 0.01 ng/μl of genomic DNA, respectively. Results demonstrate that the developed QRT-PCR method is suitable specifically for identification of S. salar in food ingredients based on the salmon growth hormone gene 1 (GH1). The processes used to develop the specific salmon reference gene case study are intended to serve as a model for performing quantification of Aquadvantage® GM salmon on future genetically modified (GM) fish to be commercialized.

  15. Selection of Suitable Reference Genes for Normalization of Quantitative Real-Time PCR in Cartilage Tissue Injury and Repair in Rabbits

    PubMed Central

    Peng, Xiao-Xiang; Zhao, Rong-Lan; Song, Wei; Chu, Hai-Rong; Li, Meng; Song, Shu-Ya; Li, Guang-Zhou; Liang, Dong-Chun

    2012-01-01

    When studying the altered expression of genes associated with cartilage regeneration by quantitative real-time RT-PCR (RT-qPCR), reference genes with highly stable expression during different stages of chondrocyte developmental are necessary to normalize gene expression accurately. Until now, no reports evaluating expression changes of commonly used reference genes in rabbit articular cartilage have been published. In this study, defects were made in rabbit articular cartilage, with or without insulin-like growth factor 1 (IGF-1) treatment, to create different chondrocyte living environments. The stability and intensity of the expressions of the candidate reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S Ribosomal RNA (18S rRNA), cyclophilin (CYP), hypoxanthine phosphoribosyl transferase (HPRT1), and β-2-microglobulin (B2M) were evaluated. The data were analyzed by geNorm and NormFinder. B2M and 18S rRNA were identified to be suitable reference genes for rabbit cartilage tissues. PMID:23203068

  16. Quantification of differences in the effective atomic numbers of healthy and cancerous tissues: A discussion in the context of diagnostics and dosimetry

    SciTech Connect

    Taylor, M. L.

    2012-09-15

    Purpose: There are a range of genetic and nongenetic factors influencing the elemental composition of different human tissues. The elemental composition of cancerous tissues frequently differs from healthy tissue of the same organ, particularly in high-Z trace element concentrations. For this reason, one could suggest that this may be exploited in diagnostics and perhaps even influence dosimetry. Methods: In this work, for the first time, effective atomic numbers are computed for common cancerous and healthy tissues using a robust, energy-dependent approach between 10 keV and 100 MeV. These are then quantitatively compared within the context of diagnostics and dosimetry. Results: Differences between effective atomic numbers of healthy and diseased tissues are found to be typically less than 10%. Fibrotic tissues and calcifications of the breast exhibit substantial (tens to hundreds of percent) differences to healthy tissue. Expectedly, differences are most pronounced in the photoelectric regime and consequently most relevant for kV imaging/therapy and radionuclides with prominent low-energy peaks. Cancerous tissue of the testes and stomach have lower effective atomic numbers than corresponding healthy tissues, while diseased tissues of the other organ sites typically have higher values. Conclusions: As dose calculation approaches improve in accuracy, there may be an argument for the explicit inclusion of pathologies. This is more the case for breast, penile, prostate, nasopharyngeal, and stomach cancer, less so for testicular and kidney cancer. The calculated data suggest dual-energy computed tomography could potentially improve lesion identification in the aforementioned organs (with the exception of testicular cancer), with most import in breast imaging. Ultimately, however, the differences are very small. It is likely that the assumption of a generic 'tissue ramp' in planning will be sufficient for the foreseeable future, and that the Z differences do not

  17. Proteome reference maps of vegetative tissues in pea. An investigation of nitrogen mobilization from leaves during seed filling.

    PubMed

    Schiltz, Séverine; Gallardo, Karine; Huart, Myriam; Negroni, Luc; Sommerer, Nicolas; Burstin, Judith

    2004-08-01

    A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen.

  18. Proteome Reference Maps of Vegetative Tissues in Pea. An Investigation of Nitrogen Mobilization from Leaves during Seed Filling1

    PubMed Central

    Schiltz, Séverine; Gallardo, Karine; Huart, Myriam; Negroni, Luc; Sommerer, Nicolas; Burstin, Judith

    2004-01-01

    A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen. PMID:15299134

  19. Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions.

    PubMed

    Imai, Tsuyoshi; Ubi, Benjamin E; Saito, Takanori; Moriguchi, Takaya

    2014-01-01

    We have evaluated suitable reference genes for real time (RT)-quantitative PCR (qPCR) analysis in Japanese pear (Pyrus pyrifolia). We tested most frequently used genes in the literature such as β-Tubulin, Histone H3, Actin, Elongation factor-1α, Glyceraldehyde-3-phosphate dehydrogenase, together with newly added genes Annexin, SAND and TIP41. A total of 17 primer combinations for these eight genes were evaluated using cDNAs synthesized from 16 tissue samples from four groups, namely: flower bud, flower organ, fruit flesh and fruit skin. Gene expression stabilities were analyzed using geNorm and NormFinder software packages or by ΔCt method. geNorm analysis indicated three best performing genes as being sufficient for reliable normalization of RT-qPCR data. Suitable reference genes were different among sample groups, suggesting the importance of validation of gene expression stability of reference genes in the samples of interest. Ranking of stability was basically similar between geNorm and NormFinder, suggesting usefulness of these programs based on different algorithms. ΔCt method suggested somewhat different results in some groups such as flower organ or fruit skin; though the overall results were in good correlation with geNorm or NormFinder. Gene expression of two cold-inducible genes PpCBF2 and PpCBF4 were quantified using the three most and the three least stable reference genes suggested by geNorm. Although normalized quantities were different between them, the relative quantities within a group of samples were similar even when the least stable reference genes were used. Our data suggested that using the geometric mean value of three reference genes for normalization is quite a reliable approach to evaluating gene expression by RT-qPCR. We propose that the initial evaluation of gene expression stability by ΔCt method, and subsequent evaluation by geNorm or NormFinder for limited number of superior gene candidates will be a practical way of finding out

  20. Quantification of the humoral immune response and hemoplasma blood and tissue loads in cats coinfected with 'Candidatus Mycoplasma haemominutum' and feline leukemia virus.

    PubMed

    Wolf-Jäckel, Godelind A; Cattori, Valentino; Geret, Catrina P; Novacco, Marilisa; Meli, Marina L; Riond, Barbara; Boretti, Felicitas S; Lutz, Hans; Hofmann-Lehmann, Regina

    2012-08-01

    'Candidatus Mycoplasma haemominutum' (CMhm) is a hemotropic mycoplasma (aka hemoplasma) of domestic cats and wild felids. In a transmission study, we exposed eight specified pathogen-free cats to blood from Iberian lynxes (Lynx pardinus) infected with CMhm. The cats were coinfected with feline leukemia virus (FeLV) from an Iberian lynx or with a prototype FeLV. The goal of the present study was to quantify the humoral immune response to CMhm and to identify potential target tissues and sequestration sites. Antibodies were measured by a recombinant antigen-based enzyme-linked immunosorbent assay, and blood and tissue loads were quantified using real-time PCR. Seven out of eight cats became CMhm-infected; all of these cats seroconverted between 3 and 13 weeks after inoculation. Antibody levels correlated with the CMhm blood loads. The peak CMhm blood loads were inversely correlated with the incubation period. PCR-positive results were found in all 24 tissues tested but not for all samples. Although all tissues were PCR-positive in one cat euthanized ten weeks after infection, many tissues tested negative in six cats euthanized at week 20 after infection. In several cats, the spleen, lung, liver, heart and aorta contained more copies than expected given the tissue's blood supply, but most tissues contained fewer copies than expected. In conclusion, this is the first study to quantify the humoral immune response and tissue loads in CMhm-FeLV-coinfected cats. The tissue loads appeared to correlate with the duration of infection and with the blood loads, but no evidence of significant CMhm tissue sequestration was found.

  1. Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with (13)C-encoded natural S1P as internal standard.

    PubMed

    Mirzaian, Mina; Wisse, Patrick; Ferraz, Maria J; Marques, André R A; Gabriel, Tanit L; van Roomen, Cindy P A A; Ottenhoff, Roelof; van Eijk, Marco; Codée, Jeroen D C; van der Marel, Gijsbert A; Overkleeft, Herman S; Aerts, Johannes M

    2016-08-01

    We developed a mass spectrometric procedure to quantify sphingosine-1-phosphate (S1P) in biological materials. The use of newly synthesized (13)C5 C18-S1P and commercial C17-S1P as internal standards rendered very similar results with respect to linearity, limit of detection and limit of quantitation. Caution is warranted with determination of plasma S1P levels. Earlier it was reported that S1P is elevated in plasma of Fabry disease patients. We investigated this with the improved quantification. No clear conclusion could be drawn for patient plasma samples given the lack of uniformity of blood collection and plasma preparation. To still obtain insight, plasma and tissues were identically collected from α-galactosidase A deficient Fabry mice and matched control animals. No significant difference was observed in plasma S1P levels. A significant 2.3 fold increase was observed in kidney of Fabry mice, but not in liver and heart. Comparative analysis of S1P in cultured fibroblasts from normal subjects and classically affected Fabry disease males revealed no significant difference. In conclusion, accurate quantification of S1P in biological materials is feasible by mass spectrometry using the internal standards (13)C5 C18-S1P or C17-S1P. Significant local increases of S1P in the kidney might occur in Fabry disease as suggested by the mouse model.

  2. Detection of West Nile virus using formalin fixed paraffin embedded tissues in crows and horses: quantification of viral transcripts by real-time RT-PCR.

    PubMed

    Tewari, Deepanker; Kim, Hyun; Feria, Willard; Russo, Brigite; Acland, Helen

    2004-08-01

    West Nile virus (WNV) RNA was quantified in WNV infected crows and horses with the help of a real-time reverse transcriptase-PCR assay. A 5' nuclease assay, based on NS5 gene detection with a fluorescent probe was used for quantifying WNV RNA using formalin fixed paraffin embedded tissue specimens. Quantitative detection of WNV RNA showed the presence of a higher amount of the viral RNA in crow tissues compared to equine tissues and these results correlated well with the detection of WNV antigen by immunostaining. In crows, the highest amount of virus was seen in the intestine and in horses in the brain.

  3. Double stable isotope ultra performance liquid chromatographic-tandem mass spectrometric quantification of tissue content and activity of phenylethanolamine N-methyltransferase, the crucial enzyme responsible for synthesis of epinephrine.

    PubMed

    Qin, Nan; Peitzsch, Mirko; Menschikowski, Mario; Siegert, Gabriele; Pacak, Karel; Eisenhofer, Graeme

    2013-02-01

    Here, we describe a novel method utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for synthesis of the stress hormone, epinephrine. The method is based on measurement of deuterium-labeled epinephrine produced from the reaction of norepinephrine with deuterium-labeled S-adenosyl-L-methionine as the methyl donor. In addition to enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of deuterium-labeled epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n = 10) and 10.9 to 13.6 % (n = 10). We established utility of the method by showing induction of the enzyme by dexamethasone in mouse pheochromocytoma cells and strong relationships to PNMT gene expression and tissue epinephrine levels in human pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final enzyme responsible for synthesis of epinephrine, the primary fight-or-flight stress hormone.

  4. Immunohistochemical quantification of the cobalamin transport protein, cell surface receptor and Ki-67 in naturally occurring canine and feline malignant tumors and in adjacent normal tissues

    PubMed Central

    Sysel, Annette M.; Valli, Victor E.; Bauer, Joseph A.

    2015-01-01

    Cancer cells have an obligate need for cobalamin (vitamin B12) to enable DNA synthesis necessary for cellular replication. This study quantified the immunohistochemical expression of the cobalamin transport protein (transcobalamin II; TCII), cell surface receptor (transcobalamin II-R; TCII-R) and proliferation protein (Ki-67) in naturally occurring canine and feline malignant tumors, and compared these results to expression in corresponding adjacent normal tissues. All malignant tumor tissues stained positively for TCII, TCII-R and Ki-67 proteins; expression varied both within and between tumor types. Expression of TCII, TCII-R and Ki-67 was significantly higher in malignant tumor tissues than in corresponding adjacent normal tissues in both species. There was a strong correlation between TCII and TCII-R expression, and a modest correlation between TCII-R and Ki-67 expression in both species; a modest association between TCII and Ki-67 expression was present in canine tissues only. These results demonstrate a quantifiable, synchronous up-regulation of TCII and TCII-R expression by proliferating canine and feline malignant tumors. The potential to utilize these proteins as biomarkers to identify neoplastic tissues, streamline therapeutic options, evaluate response to anti-tumor therapy and monitor for recurrent disease has important implications in the advancement of cancer management for both human and companion animal patients. PMID:25633912

  5. Dynamic OCT monitoring and quantification of light penetration enhancement for normal, benign and cancerous human lung tissues at different concentrations of glycerol

    SciTech Connect

    Shu-wen Tan; Ying Jin; Hui Yu; Guo-yong Wu

    2013-10-31

    We have evaluated the dynamic effects of the analyte diffusion on the 1/e light penetration depths of normal, benign and cancerous human lung tissue in vitro, as well as have monitored and quantified the dynamic change in the light penetration depths of the mentioned human lung tissue after application of 25 % and 50 % glycerol solution, respectively. The light penetration depths of the analyte diffusion in the lung tissue are measured using the Fourierdomain optical coherence tomography (FD-OCT). Experimental results show that the application of glycerol as a chemical agent can significantly enhance light penetration depths into the human normal lung (NL), lung benign granulomatosis (LBG) and lung squamous cell carcinoma (LSCC) tissue. In-depth transport of the glycerol molecules in the NL, LBG and LSCC tissue at a lower glycerol concentration (25 %) are faster than those at a higher glycerol concentration (50 %), and the 1/e light penetration depths at a lower glycerol concentration (25 %) are smaller than those at a higher glycerol concentration (50 %), respectively. Their differences in the maximal 1/e light penetration depths of the NL, LBG and LSCC tissue at a higher and a lower glycerol concentrations were only 8.8 %, 6.8 % and 4.7 %, respectively. (biophotonics)

  6. Proteomic analysis of breast cancer tissues to identify biomarker candidates by gel-assisted digestion and label-free quantification methods using LC-MS/MS.

    PubMed

    Song, Mi-Na; Moon, Pyong-Gon; Lee, Jeong-Eun; Na, MinKyun; Kang, Wonku; Chae, Yee Soo; Park, Ji-Young; Park, Hoyong; Baek, Moon-Chang

    2012-10-01

    This study presents a proteomic method that differentiates between matched normal and breast tumor tissues from ductal carcinoma in situ (DCIS) and invasive carcinoma from Korean women, to identify biomarker candidates and to understand pathogenesis of breast cancer in protein level. Proteins from tissues obtained by biopsy were extracted by RIPA buffer, digested by the gel-assisted method, and analyzed by nano-UPLC-MS/MS. From proteomic analysis based on label-free quantitation strategy, a non-redundant list of 298 proteins was identified from the normal and tumor tissues, and 244 proteins were quantified using IDEAL-Q software. Hierarchical clustering analysis showed two patterns classified as two groups, invasive carcinoma and DCIS, suggesting a difference between two carcinoma at the protein expression level as expected. Differentially expressed proteins in tumor tissues compared to the corresponding normal tissues were related to three biological pathways: antigen-processing and presentation, glycolysis/gluconeogenesis, and complement and coagulation cascades. Among them, the up-regulation of calreticulin (CRT) and protein disulfide isomerase A3 (PDIA3) was confirmed by Western blot analysis. In conclusion, this study showed the possibility of identifying biomarker candidates for breast cancer using tissues and might help to understand the pathophysiology of this cancer at the protein level.

  7. Heat treatment and the use of additives to improve the stability of paralytic shellfish poisoning toxins in shellfish tissue reference materials for internal quality control and proficiency testing.

    PubMed

    Burrell, Stephen; Clion, Valentin; Auroy, Virginie; Foley, Barry; Turner, Andrew D

    2015-06-01

    The need for homogenous reference materials stable for paralytic shellfish toxins is vital for the monitoring and quality assurance of these potent neurotoxins in shellfish. Two stabilisation techniques were investigated, heat treatment through autoclaving and the addition of preserving additives into the tissue matrix. Short and long-term stability experiments as well as homogeneity determination were conducted on materials prepared by both techniques in comparison with an untreated control using two LC-FLD methods. Both techniques improved the stability of the matrix and the PSP toxins present compared to the controls. A material was prepared using the combined techniques of heat treatment followed by spiking with additives and data is presented from this optimised reference material as used over a two year period in the Irish national monitoring program and in a development exercise as part of a proficiency testing scheme operated by QUASIMEME (Quality Assurance of Information for Marine Environmental Monitoring in Europe) since 2011. The results were indicative of the long-term stability of the material as evidenced through consistent assigned values in the case of the proficiency testing scheme and a low relative standard deviation of 10.5% for total toxicity data generated over 24 months.

  8. Direct 4D PET MLEM reconstruction of parametric images using the simplified reference tissue model with the basis function method for [¹¹C]raclopride.

    PubMed

    Gravel, Paul; Reader, Andrew J

    2015-06-07

    This work assesses the one-step late maximum likelihood expectation maximization (OSL-MLEM) 4D PET reconstruction algorithm for direct estimation of parametric images from raw PET data when using the simplified reference tissue model with the basis function method (SRTM-BFM) for the kinetic analysis. To date, the OSL-MLEM method has been evaluated using kinetic models based on two-tissue compartments with an irreversible component. We extend the evaluation of this method for two-tissue compartments with a reversible component, using SRTM-BFM on simulated 3D + time data sets (with use of [(11)C]raclopride time-activity curves from real data) and on real data sets acquired with the high resolution research tomograph. The performance of the proposed method is evaluated by comparing voxel-level binding potential (BPND) estimates with those obtained from conventional post-reconstruction kinetic parameter estimation. For the commonly chosen number of iterations used in practice, our results show that for the 3D + time simulation, the direct method delivers results with lower (%)RMSE at the normal count level (decreases of 9-10 percentage points, corresponding to a 38-44% reduction), and also at low count levels (decreases of 17-21 percentage points, corresponding to a 26-36% reduction). As for the real 3D data set, the results obtained follow a similar trend, with the direct reconstruction method offering a 21% decrease in (%)CV compared to the post reconstruction method at low count levels. Thus, based on the results presented herein, using the SRTM-BFM kinetic model in conjunction with the OSL-MLEM direct 4D PET MLEM reconstruction method offers an improvement in performance when compared to conventional post reconstruction methods.

  9. Evaluating a set of reference genes for expression normalization in multiple tissues and skeletal muscle at different development stages in pigs using quantitative real-time polymerase chain reaction.

    PubMed

    Zhang, Jing; Tang, Zhonglin; Wang, Ning; Long, Liangqi; Li, Kui

    2012-01-01

    Gene expression analysis requires the use of reference genes consistently expressed under various conditions. In many cases, however, the commonly used reference genes are not uniformly expressed independently of tissues or environmental conditions. To provide a set of reliable reference genes in pigs, we used quantitative polymerase chain reaction to examine expression of six common reference genes (GAPDH, ACTB, H3F3A, HPRT1, RPL32, and RPS18) in adult tissues and prenatal skeletal muscles at 33, 65, and 90 days postcopulation from Tongcheng (obese-type) and Landrace (lean-type) pigs. The expression stability of these reference genes was evaluated by NormFinder, BestKeeper, and geNorm methods. Our data suggest that the reference genes were expressed variably in different tissues, developmental stages and breeds. RPS18, PRL32, and H3F3A could be used as internal controls to normalize gene expression in pig tissues and developmental skeletal muscle. The combination of internal control genes was necessary for accurate expression normalization. During skeletal muscle development, H3F3A and RPS18 would be the most appropriate combination to normalize gene expression in Tongcheng pigs, whereas the combination of PRL32 and RPS18 would be more suitable in Landrace pigs. In different tissues, the expression of PRL32 and RPS18 was the most consistent, and the combination of three genes (RPL32, RPS18, and H3F3A) is the most suitable for accurate normalization.

  10. Simultaneous Quantification of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Rats Shows Strong Correlations between Serum and Brain Tissue Levels.

    PubMed

    Xue, Ying; He, Xin; Li, Huan-De; Deng, Yang; Yan, Miao; Cai, Hua-Lin; Tang, Mi-Mi; Dang, Rui-Li; Jiang, Pei

    2015-01-01

    While vitamin D3 is recognized as a neuroactive steroid affecting both brain development and function, efficient analytical method in determining vitamin D3 metabolites in the brain tissue is still lacking, and the relationship of vitamin D3 status between serum and brain remains elusive. Therefore, we developed a novel analysis method by using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to simultaneously quantify the concentrations of 25-hydroxyvitamin D3 (25(OH)D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in the serum and brain of rats fed with different dose of vitamin D3. We further investigated whether variations of serum vitamin D3 metabolites could affect vitamin D3 metabolite levels in the brain. Serum and brain tissue were analyzed by HPLC-MS/MS with electrospray ionization following derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). The method is highly sensitive, specific, and accurate to quantify 25(OH)D3 and 24,25(OH)2D3 in animal brain tissue. Vitamin D3 metabolites in brain tissue were significantly lower in rats fed with a vitamin D deficiency diet than in rats fed with high vitamin D3 diet. There was also a strong correlation of vitamin D3 metabolites in serum and brain. These results indicate that vitamin D3 status in serum affects bioavailability of vitamin D3 metabolites in the brain.

  11. Simultaneous Quantification of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Rats Shows Strong Correlations between Serum and Brain Tissue Levels

    PubMed Central

    Xue, Ying; He, Xin; Li, Huan-De; Deng, Yang; Yan, Miao; Cai, Hua-Lin; Tang, Mi-Mi; Dang, Rui-Li; Jiang, Pei

    2015-01-01

    While vitamin D3 is recognized as a neuroactive steroid affecting both brain development and function, efficient analytical method in determining vitamin D3 metabolites in the brain tissue is still lacking, and the relationship of vitamin D3 status between serum and brain remains elusive. Therefore, we developed a novel analysis method by using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to simultaneously quantify the concentrations of 25-hydroxyvitamin D3 (25(OH)D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in the serum and brain of rats fed with different dose of vitamin D3. We further investigated whether variations of serum vitamin D3 metabolites could affect vitamin D3 metabolite levels in the brain. Serum and brain tissue were analyzed by HPLC-MS/MS with electrospray ionization following derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). The method is highly sensitive, specific, and accurate to quantify 25(OH)D3 and 24,25(OH)2D3 in animal brain tissue. Vitamin D3 metabolites in brain tissue were significantly lower in rats fed with a vitamin D deficiency diet than in rats fed with high vitamin D3 diet. There was also a strong correlation of vitamin D3 metabolites in serum and brain. These results indicate that vitamin D3 status in serum affects bioavailability of vitamin D3 metabolites in the brain. PMID:26713090

  12. Real-time quantification of proteins secreted by artificial connective tissue made from uni- or multidirectional collagen I scaffolds and oral mucosa fibroblasts.

    PubMed

    Bustos, Rosa Helena; Suesca, Edward; Millán, Diana; González, José Manuel; Fontanilla, Marta R

    2014-03-04

    Previously, we found that oral autologous artificial connective tissue (AACT) had a different protein secretion profile to that of clot-embedded AACT. Other oral mucosa substitutes, having different cell types and scaffolds, had dissimilar secretion profiles of proteins (including that for AACT) that influence healing outcome; thus, to ascertain the profiles of factors secreted by artificial tissue and whether they are influenced by their microstructure might help in understanding their bioactivity. An important component of tissue microstructure is the fiber orientation of the scaffold used for manufacturing it. This work developed a surface plasmon resonance (SPR) methodology to quantify factors secreted by oral artificial connective tissue (ACT) in culture medium, and a method to manufacture unidirectional laminar collagen I scaffolds. The SPR methodology was used for assessing differences in the protein secretion profile of ACT made with collagen scaffolds having different fiber orientation (unidirectional vs multidirectional). Oral fibroblasts seeded onto unidirectional scaffolds increased the secretion of six factors involved in modulating healing compared to those seeded onto multidirectional scaffolds. Histological analysis of uni- and multidirectional ACT showed that cells differ in their alignment and morphology. This SPR-methodology led to nanoscale detection of paracrine factors and might be useful to study biomarkers of three-dimensional cell growth, cell differentiation, and wound-healing progression.

  13. Concurrent quantification of tissue metabolism and blood flow via 2H/31P NMR in vivo. III. Alterations of muscle blood flow and metabolism during sepsis.

    PubMed

    Song, S K; Hotchkiss, R S; Karl, I E; Ackerman, J J

    1992-05-01

    In the conclusion of this series of reports, the application of 31P/2H NMR to investigate the pathophysiology of sepsis in rat hindlimb muscle is demonstrated. Sepsis decreased muscle [PCr] by 18%, 18 +/- 4 SD vs 22 +/- 4 SD mmol/kg tissue wet wt (P = 0.01) in control rats but [ATP] was unchanged, 6 mmol/kg tissue wet wt (P = 0.2). The derived free cytosolic [ADP] in the two groups was similar, [ADP]septic = 0.023 +/- 0.004 SD and [ADP]control = 0.021 +/- 0.003 SD mmol/kg tissue wet wt, and not statistically different (P = 0.14). Likewise [Pi] in the septic and control groups was not statistically different, [Pi]septic = 1.1 +/- 0.5 SD and [Pi]control = 1.2 +/- 0.4 SD mmol/kg tissue wet wt (P = 0.2). Septic rats presented the symptom of respiratory alkalosis evidenced by elevated blood pH. Sepsis decreased muscle blood flow by 33%, P = 0.003, but examination of individual subjects did not demonstrate a correlation with the reduction in [PCr]. Thus, a metabolic energy deficit caused by cellular ischemia/hypoxia is not a likely cause of cellular abnormality in rat hindlimb muscle during sepsis.

  14. Using Pharmacokinetic Profiles and Digital Quantification of Stained Tissue Microarrays as a Medium-Throughput, Quantitative Method for Measuring the Kinetics of Early Signaling Changes Following Integrin-Linked Kinase Inhibition in an In Vivo Model of Cancer

    PubMed Central

    Dragowska, Weislawa H.; Bally, Marcel B.

    2015-01-01

    A small molecule inhibitor (QLT0267) targeting integrin-linked kinase is able to slow breast tumor growth in vivo; however, the mechanism of action remains unknown. Understanding how targeting molecules involved in intersecting signaling pathways impact disease is challenging. To facilitate this understanding, we used tumor tissue microarrays (TMA) and digital image analysis for quantification of immunohistochemistry (IHC) in order to investigate how QLT0267 affects signaling pathways in an orthotopic model of breast cancer over time. Female NCR nude mice were inoculated with luciferase-positive human breast tumor cells (LCC6Luc) and tumor growth was assessed by bioluminescent imaging (BLI). The plasma levels of QLT0267 were determined by LC-MS/MS methods following oral dosing of QLT0267 (200 mg/kg). A TMA was constructed using tumor tissue collected at 2, 4, 6, 24, 78 and 168 hr after treatment. IHC methods were used to assess changes in ILK-related signaling. The TMA was digitized, and Aperio ScanScope and ImageScope software were used to provide semi-quantitative assessments of staining levels. Using medium-throughput IHC quantitation, we show that ILK targeting by QLT0267 in vivo influences tumor physiology through transient changes in pathways involving AKT, GSK-3 and TWIST accompanied by the translocation of the pro-apoptotic protein BAD and an increase in Caspase-3 activity. PMID:25940338

  15. Using Pharmacokinetic Profiles and Digital Quantification of Stained Tissue Microarrays as a Medium-Throughput, Quantitative Method for Measuring the Kinetics of Early Signaling Changes Following Integrin-Linked Kinase Inhibition in an In Vivo Model of Cancer.

    PubMed

    Kalra, Jessica; Dragowska, Weislawa H; Bally, Marcel B

    2015-09-01

    A small molecule inhibitor (QLT0267) targeting integrin-linked kinase is able to slow breast tumor growth in vivo; however, the mechanism of action remains unknown. Understanding how targeting molecules involved in intersecting signaling pathways impact disease is challenging. To facilitate this understanding, we used tumor tissue microarrays (TMA) and digital image analysis for quantification of immunohistochemistry (IHC) in order to investigate how QLT0267 affects signaling pathways in an orthotopic model of breast cancer over time. Female NCR nude mice were inoculated with luciferase-positive human breast tumor cells (LCC6(Luc)) and tumor growth was assessed by bioluminescent imaging (BLI). The plasma levels of QLT0267 were determined by LC-MS/MS methods following oral dosing of QLT0267 (200 mg/kg). A TMA was constructed using tumor tissue collected at 2, 4, 6, 24, 78 and 168 hr after treatment. IHC methods were used to assess changes in ILK-related signaling. The TMA was digitized, and Aperio ScanScope and ImageScope software were used to provide semi-quantitative assessments of staining levels. Using medium-throughput IHC quantitation, we show that ILK targeting by QLT0267 in vivo influences tumor physiology through transient changes in pathways involving AKT, GSK-3 and TWIST accompanied by the translocation of the pro-apoptotic protein BAD and an increase in Caspase-3 activity.

  16. Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of glucocorticoid residues in edible tissues of swine, cattle, sheep, and chicken.

    PubMed

    Chen, Dongmei; Tao, Yanfei; Liu, Zhaoying; Liu, Zhenli; Wang, Yulian; Huang, Lingli; Yuan, Zonghui

    2010-10-01

    A confirmatory and quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the presence of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, and fludrocortisone) in the muscles and livers of swine, cattle, and sheep and the muscle of chicken is described. After deconjugation in alkali media, samples were extracted with ethyl acetate for glucocorticoids followed by solid-phase extraction clean-up and reconstitution in the LC mobile phase. The hydrolysis procedure with sodium hydroxide was used to reduce handling time. A single-step solid-phase extraction method was optimized which is suitable for the clean-up of the compounds of interest in many diverse tissue matrices. LC separations were performed on a C(18) column with gradient elution using acetonitrile and water (containing 0.2% formic acid) and the two epimers betamethasone and dexamethasone were successfully separated. LC-electrospray ionization (ESI)-MS/MS in negative mode with selected reaction monitoring (SRM) mode was performed to improve method sensitivity and reduce matrix interference. Two SRM transitions were used for each compound. The recovery of glucocorticoids spiked at levels of 0.5-16 microg kg(-1) ranged from 55% to 107%; the between-day relative standard deviations were no more than 15%. The limits of quantification were 0.5-2.0 microg kg(-1) in muscle and 1-4 microg kg(-1) in liver. The optimized procedure was successfully applied to monitor the food at the 2008 Summer Olympics Games in Beijing, China, demonstrating the method to be simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.

  17. On the use of 31P NMR for the quantification of hydrosoluble phosphorus-containing compounds in coral host tissues and cultured zooxanthellae

    NASA Astrophysics Data System (ADS)

    Godinot, Claire; Gaysinski, Marc; Thomas, Olivier P.; Ferrier-Pagès, Christine; Grover, Renaud

    2016-02-01

    31P Nuclear Magnetic Resonance (NMR) was assessed to investigate the phosphorus-containing compounds present in the tissues of the scleractinian coral Stylophora pistillata as well as of cultured zooxanthellae (CZ). Results showed that phosphorus-containing compounds observed in CZ were mainly phosphate and phosphate esters. Phosphate accounted for 19 ± 2% of the total phosphorus compounds observed in CZ maintained under low P-levels (0.02 μM). Adding 5 mM of dissolved inorganic phosphorus (KH2PO4) to the CZ culture medium led to a 3.1-fold increase in intracellular phosphate, while adding 5 mM of dissolved organic phosphorus led to a reduction in the concentration of phosphorus compounds, including a 2.5-fold intracellular phosphate decrease. In sharp contrast to zooxanthellae, the host mainly contained phosphonates, and to a lesser extent, phosphate esters and phosphate. Two-months of host starvation decreased the phosphate content by 2.4 fold, while bleaching of fed corals did not modify this content. Based on 31P NMR analyses, this study highlights the importance of phosphonates in the composition of coral host tissues, and illustrates the impact of phosphorus availability on the phosphorus composition of host tissues and CZ, both through feeding of the host and inorganic phosphorus enrichment of the CZ.

  18. Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi's sarcoma

    PubMed Central

    2010-01-01

    In Cuba, previous reports have shown an increase of epidemic KS, reaching a total of 120 cases by the end of 2007, despite the use of HAART. To evaluate and compare the role of human herpes virus 8 (HHV-8) viral loads in different compartments of AIDS-related Kaposi's sarcoma (AIDS-KS) patients real-time polymerase chain reaction (RT-PCR) was used to determine the genome copy number of HHV-8 in plasma, saliva, tissue and peripheral blood mononuclear cells (PBMC) of 49 AIDS-KS patients. Overall, 98% of AIDS-KS patients harbored detectable HHV-8. HHV-8 could be detected in 91.6% of KS tissue lesions showing the highest viral load (median log = 3.14 copies/100 ng DNA) followed by saliva and PBMC which were positive in 78%, and 69.2%; respectively. In contrast, HHV-8 was detected in only 37% of plasma samples, which also showed lower viral loads. Men who had sex with men (MSM) were more likely to have three-times higher HHV-8 genome copies in KS lesions when compared with tissues from heterosexuals individuals (OR 3; 95% CI 1.1 to 12.5). These results emphasize the systemic nature of HHV-8-infection and demonstrate the possible role of saliva in HHV-8 transmission among MSM. PMID:21429238

  19. On the use of 31P NMR for the quantification of hydrosoluble phosphorus-containing compounds in coral host tissues and cultured zooxanthellae.

    PubMed

    Godinot, Claire; Gaysinski, Marc; Thomas, Olivier P; Ferrier-Pagès, Christine; Grover, Renaud

    2016-02-23

    (31)P Nuclear Magnetic Resonance (NMR) was assessed to investigate the phosphorus-containing compounds present in the tissues of the scleractinian coral Stylophora pistillata as well as of cultured zooxanthellae (CZ). Results showed that phosphorus-containing compounds observed in CZ were mainly phosphate and phosphate esters. Phosphate accounted for 19 ± 2% of the total phosphorus compounds observed in CZ maintained under low P-levels (0.02 μM). Adding 5 mM of dissolved inorganic phosphorus (KH2PO4) to the CZ culture medium led to a 3.1-fold increase in intracellular phosphate, while adding 5 mM of dissolved organic phosphorus led to a reduction in the concentration of phosphorus compounds, including a 2.5-fold intracellular phosphate decrease. In sharp contrast to zooxanthellae, the host mainly contained phosphonates, and to a lesser extent, phosphate esters and phosphate. Two-months of host starvation decreased the phosphate content by 2.4 fold, while bleaching of fed corals did not modify this content. Based on (31)P NMR analyses, this study highlights the importance of phosphonates in the composition of coral host tissues, and illustrates the impact of phosphorus availability on the phosphorus composition of host tissues and CZ, both through feeding of the host and inorganic phosphorus enrichment of the CZ.

  20. On the use of 31P NMR for the quantification of hydrosoluble phosphorus-containing compounds in coral host tissues and cultured zooxanthellae

    PubMed Central

    Godinot, Claire; Gaysinski, Marc; Thomas, Olivier P.; Ferrier-Pagès, Christine; Grover, Renaud

    2016-01-01

    31P Nuclear Magnetic Resonance (NMR) was assessed to investigate the phosphorus-containing compounds present in the tissues of the scleractinian coral Stylophora pistillata as well as of cultured zooxanthellae (CZ). Results showed that phosphorus-containing compounds observed in CZ were mainly phosphate and phosphate esters. Phosphate accounted for 19 ± 2% of the total phosphorus compounds observed in CZ maintained under low P-levels (0.02 μM). Adding 5 mM of dissolved inorganic phosphorus (KH2PO4) to the CZ culture medium led to a 3.1-fold increase in intracellular phosphate, while adding 5 mM of dissolved organic phosphorus led to a reduction in the concentration of phosphorus compounds, including a 2.5-fold intracellular phosphate decrease. In sharp contrast to zooxanthellae, the host mainly contained phosphonates, and to a lesser extent, phosphate esters and phosphate. Two-months of host starvation decreased the phosphate content by 2.4 fold, while bleaching of fed corals did not modify this content. Based on 31P NMR analyses, this study highlights the importance of phosphonates in the composition of coral host tissues, and illustrates the impact of phosphorus availability on the phosphorus composition of host tissues and CZ, both through feeding of the host and inorganic phosphorus enrichment of the CZ. PMID:26902733

  1. A pseudo-three-dimensional model for quantification of oxygen diffusion from pre-glomerular arteries to renal tissue and renal venous blood.

    PubMed

    Lee, Chang-Joon; Ngo, Jennifer P; Kar, Saptarshi; Gardiner, Bruce S; Evans, Roger G; Smith, David W

    2017-04-05

    To assess the physiological significance of arterial-to-venous (AV) oxygen shunting, we generated a new pseudo-three-dimensional computational model of oxygen diffusion from intrarenal arteries to cortical tissue and veins. The model combines the eleven branching levels (known as 'Strahler' orders) of the pre-glomerular renal vasculature in the rat, with an analysis of an extensive dataset obtained using light microscopy to estimate oxygen mass transfer coefficients for each Strahler order. Further, the AV shunting model is now set within a global oxygen transport model that includes transport from arteries, glomeruli, peritubular capillaries and veins to tissue. While a number of lines of evidence suggest AV shunting is significant, most importantly our AV oxygen shunting model predicts AV shunting is small under normal physiological conditions (~0.9% of total renal oxygen delivery; range 0.4% to 1.4%), but increases during renal ischemia, glomerular hyperfiltration (~2.1% of total renal oxygen delivery; range 0.84% to 3.36%) and some cardiovascular disease states (~3.0% of total renal oxygen delivery; range 1.2% to 4.8%). Under normal physiological conditions, blood PO2 is predicted to fall by ~16 mmHg from the root of the renal artery to glomerular entry, with AV oxygen shunting contributing ~40% and oxygen diffusion from arteries to tissue contributing ~60% of this decline. Arterial PO2 is predicted to fall most rapidly from Strahler order 4, under normal physiological conditions. We conclude that AV oxygen shunting normally has only a small impact on renal oxygenation, but may exacerbate renal hypoxia during renal ischemia, hyperfiltration and some cardiovascular disease states.

  2. Protocols for staining of bile canalicular and sinusoidal networks of human, mouse and pig livers, three-dimensional reconstruction and quantification of tissue microarchitecture by image processing and analysis.

    PubMed

    Hammad, Seddik; Hoehme, Stefan; Friebel, Adrian; von Recklinghausen, Iris; Othman, Amnah; Begher-Tibbe, Brigitte; Reif, Raymond; Godoy, Patricio; Johann, Tim; Vartak, Amruta; Golka, Klaus; Bucur, Petru O; Vibert, Eric; Marchan, Rosemarie; Christ, Bruno; Dooley, Steven; Meyer, Christoph; Ilkavets, Iryna; Dahmen, Uta; Dirsch, Olaf; Böttger, Jan; Gebhardt, Rolf; Drasdo, Dirk; Hengstler, Jan G

    2014-05-01

    Histological alterations often constitute a fingerprint of toxicity and diseases. The extent to which these alterations are cause or consequence of compromised organ function, and the underlying mechanisms involved is a matter of intensive research. In particular, liver disease is often associated with altered tissue microarchitecture, which in turn may compromise perfusion and functionality. Research in this field requires the development and orchestration of new techniques into standardized processing pipelines that can be used to reproducibly quantify tissue architecture. Major bottlenecks include the lack of robust staining, and adequate reconstruction and quantification techniques. To bridge this gap, we established protocols employing specific antibody combinations for immunostaining, confocal imaging, three-dimensional reconstruction of approximately 100-μm-thick tissue blocks and quantification of key architectural features. We describe a standard procedure termed 'liver architectural staining' for the simultaneous visualization of bile canaliculi, sinusoidal endothelial cells, glutamine synthetase (GS) for the identification of central veins, and DAPI as a nuclear marker. Additionally, we present a second standard procedure entitled 'S-phase staining', where S-phase-positive and S-phase-negative nuclei (stained with BrdU and DAPI, respectively), sinusoidal endothelial cells and GS are stained. The techniques include three-dimensional reconstruction of the sinusoidal and bile canalicular networks from the same tissue block, and robust capture of position, size and shape of individual hepatocytes, as well as entire lobules from the same tissue specimen. In addition to the protocols, we have also established image analysis software that allows relational and hierarchical quantifications of different liver substructures (e.g. cells and vascular branches) and events (e.g. cell proliferation and death). Typical results acquired for routinely quantified

  3. The expression of genes PKM2 and CAST in the muscle tissue of pigs differentiated by glycolytic potential and drip loss, with reference to the genetic group.

    PubMed

    Sieczkowska, H; Zybert, A; Krzecio, E; Antosik, K; Koćwin-Podsiadła, M; Pierzchała, M; Urbański, P

    2010-01-01

    The present studies aimed at an analysis of the expression level of genes PKM2 and CAST in Longissimus lumborum [LL] muscle tissue of pigs differing as regards the glycolytic potential [GP] and drip loss [DL] from the LL muscle, with reference to the genetic group. The studies covered a total of 65 pigs: 20 purebred Landrace [L], 22 crossbreeds of Landrace with the Yorkshire [L x Y] and 23 three-breed crosses (Landrace x Yorkshire) x Duroc [(L x Y) x D]. In the case of gene PKM2 one may observe in (L x Y) x D crossbreds, compared to L x Y crossbreds, an increased expression, closely related with the increase in dry matter content, including intramuscular fat, as well as a more favourable progress of glycolytic and energy metabolism during the early time post mortem (pH(45) and R(1)). Compared with Landrace animals, the lower expression of the CAST gene observed in (L x Y) x D pigs is manifested by a marked improvement of meat quality (R(1) pH(45) pH(24), pH(48)), arising from the rate of glycolytic and energy metabolism, typical for normal meat, that in effect results in its higher culinary and technological value.

  4. High-performance liquid chromatographic methods for the analysis of human parathyroid hormone in reference standards, parathyroid tissue and biological fluids.

    PubMed

    Zanelli, J M; Kent, J C; Rafferty, B; Nissenson, R A; Nice, E C; Capp, M W; O'Hare, M J

    1983-08-12

    Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to fractionate human parathyroid hormone (hPTH) from a variety of natural sources and to compare it with synthetic hPTH and hPTH fragments. Multiple radioimmunoassay systems for amino, mid and carboxyl regions of hPTH were used to monitor various preparations of hPTH previously prepared by conventional methods and ampouled in nanogram amounts for reference standard and reagent purposes. Results confirmed that they were free of detectable cleavage products, but showed that the intact hPTH comprised three or four closely associated components. A similar pattern of heterogeneity was obtained when hPTH was extracted from stored human parathyroid adenomata by a simple rapid HPLC bulk fractionation method. Comparison with synthetic 1-84 hPTH and modification of sample handling to minimize oxidative conditions, indicate that some of these components are probably intermediate oxidation products. A number of less hydrophobic components, with carboxyl region immunoreactivities, were obtained from the individual adenoma samples, human parathyroid cyst fluid, ampouled samples of human adenoma tissue culture medium, and secondary hyperparathyroid plasma ultrafiltrate when they were fractionated by RP-HPLC. The results strongly suggest that the biological degradation of hPTH is more complex than generally believed, and that RP-HPLC offers a new dimension in its analysis.

  5. Comparative Study of Metal Quantification in Neurological Tissue Using Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry Imaging and X-ray Fluorescence Microscopy.

    PubMed

    Davies, Katherine M; Hare, Dominic J; Bohic, Sylvain; James, Simon A; Billings, Jessica L; Finkelstein, David I; Doble, Philip A; Double, Kay L

    2015-07-07

    Redox-active metals in the brain mediate numerous biochemical processes and are also implicated in a number of neurodegenerative diseases. A number of different approaches are available for quantitatively measuring the spatial distribution of biometals at an image resolution approaching the subcellular level. Measured biometal levels obtained using laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS; spatial resolution 15 μm × 15 μm) were within the range of those obtained using X-ray fluorescence microscopy (XFM; spatial resolution 2 μm × 7 μm) and regional changes in metal concentration across discrete brain regions were replicated to the same degree. Both techniques are well suited to profiling changes in regional biometal distribution between healthy and diseased brain tissues, but absolute quantitation of metal levels varied significantly between methods, depending on the metal of interest. Where all possible variables affect metal levels, independent of a treatment/phenotype are controlled, either method is suitable for examining differences between experimental groups, though, as with any method for imaging post mortem brain tissue, care should be taken when interpreting the total metal levels with regard to physiological concentrations.

  6. Detection and quantification of a very high density lipoprotein in different tissues of Triatoma infestans during the last nymphal and adult stages.

    PubMed

    Rimoldi, O J; Córsico, B; González, M S; Brenner, R R

    1996-07-01

    The presence of a very high density lipoprotein (VHDL), an hexameric protein, was explored in different tissues of Triatoma infestans throughout the last nymphal and adult stages, and in egg extracts by Western blot assays. The VHDL was always detected in both, hemolymph and fat body, during the above mentioned stages and it was also observed in the buffer soluble fraction of testis and egg homogenates. An enzyme-linked immunosorbent assay (ELISA) was used to measure the VHDL titer in these tissues. Hemolymph VHDL reaches a maximum value before the last molt, then it abruptly declines in males and females just after emergence, but during adult life it increases again. Fat body VHDL decreases slowly and continuously during the nymph growth reaching a minimum value prior to molting, and in the first week of adult life the values were even two-fold lower; then, it shows a different cycle of accumulation and depletion in males and females. In adult testis the VHDL undergoes a cycle similar to the one observed in male fat body. This protein increases progressively during embryonic development and, at the time of larval hatching it reaches its maximum value. The hexameric protein presents homologies in its N-terminal sequence with storage hexamerins of Diptera, Lepidoptera and Hymenoptera.

  7. Quantification of leucocytes, T-lymphocytes and macrophages in autoptical endomyocardial tissue from 56 normal human hearts during the first year of life.

    PubMed

    Grasmeyer, Sarah; Oswald, Sylvia; Madea, Burkhard

    2016-05-01

    This study evaluated the normal number of inflammatory cells in the heart in the first year of life using two methods to compare their ability to quantitate physiological myocardial infiltration. Eight endomyocardial samples from both ventricles were obtained at autopsy from 56 structurally normal hearts during the first year of life. In each sample the numbers of leucocytes, T-lymphocytes and macrophages were counted once in 20 randomly chosen high-power fields (400×) as well as in a 10mm(2) area of randomly chosen myocardial tissue (100×) by two independent investigators. Compared to the literature a greater representative proportion of myocardial tissue was analyzed. The results of the enumeration in mm(2) were converted into high-power-fields to compare both methods. The mean numbers and standard deviations for leucocytes, T-lymphocytes and macrophages were calculated. Both counting methods showed similar results with low inflammatory cell counts per single heart and staining. A greater understanding of the physiological myocardial infiltration by leucocytes, T-lymphocytes and macrophages is important for postmortem forensic cases, and for the interpretation of endomyocardial biopsies in infants.

  8. Application of Focused Ultrasound-Assisted Extraction for the Quantification of Persistent Organic Pollutions in Liver Tissue of Giant Toad (Rhinella marina).

    PubMed

    Flores-Ramírez, R; Espinosa-Reyes, G; Cilia-López, V G; González-Mille, D J; Rodríguez-Aguilar, M; Díaz de León-Martínez, L; Díaz-Barriga, F

    2017-02-01

    A simple and rapid focused ultrasound extraction method was developed for the determination of Persistent Organic Pollutants (POPs) in liver tissue obtained of giant toad (Rhinella marina) using a gas chromatography coupled to a mass detector with electron impact ionization. The performed method for POPs, was validated in fortified matrix, showing linearity from the LOQ up to 100 ng/mL; LODs and LOQs for each compound were between 1.7 and 4.8 and 3.5-7.5 ng/mL, respectively. Recovery rates were among 79%-116% for POPs determined. Finally, the method was applied in liver samples of giant toads found in a malarial area in Mexico. The sensitivity of the proposed method was good enough to ensure reliable determination of target analytes at concentration levels commonly found in this kind of samples.

  9. A Robust Method for Transcript Quantification with RNA-Seq Data

    PubMed Central

    Huang, Yan; Hu, Yin; Jones, Corbin D.; MacLeod, James N.; Chiang, Derek Y.; Liu, Yufeng; Prins, Jan F.

    2013-01-01

    Abstract The advent of high throughput RNA-seq technology allows deep sampling of the transcriptome, making it possible to characterize both the diversity and the abundance of transcript isoforms. Accurate abundance estimation or transcript quantification of isoforms is critical for downstream differential analysis (e.g., healthy vs. diseased cells) but remains a challenging problem for several reasons. First, while various types of algorithms have been developed for abundance estimation, short reads often do not uniquely identify the transcript isoforms from which they were sampled. As a result, the quantification problem may not be identifiable, i.e., lacks a unique transcript solution even if the read maps uniquely to the reference genome. In this article, we develop a general linear model for transcript quantification that leverages reads spanning multiple splice junctions to ameliorate identifiability. Second, RNA-seq reads sampled from the transcriptome exhibit unknown position-specific and sequence-specific biases. We extend our method to simultaneously learn bias parameters during transcript quantification to improve accuracy. Third, transcript quantification is often provided with a candidate set of isoforms, not all of which are likely to be significantly expressed in a given tissue type or condition. By resolving the linear system with LASSO, our approach can infer an accurate set of dominantly expressed transcripts while existing methods tend to assign positive expression to every candidate isoform. Using simulated RNA-seq datasets, our method demonstrated better quantification accuracy and the inference of dominant set of transcripts than existing methods. The application of our method on real data experimentally demonstrated that transcript quantification is effective for differential analysis of transcriptomes. PMID:23461570

  10. Derivation and validation of simple anthropometric equations to predict adipose tissue mass and total fat mass with MRI as the reference method.

    PubMed

    Al-Gindan, Yasmin Y; Hankey, Catherine R; Govan, Lindsay; Gallagher, Dympna; Heymsfield, Steven B; Lean, Michael E J

    2015-12-14

    The reference organ-level body composition measurement method is MRI. Practical estimations of total adipose tissue mass (TATM), total adipose tissue fat mass (TATFM) and total body fat are valuable for epidemiology, but validated prediction equations based on MRI are not currently available. We aimed to derive and validate new anthropometric equations to estimate MRI-measured TATM/TATFM/total body fat and compare them with existing prediction equations using older methods. The derivation sample included 416 participants (222 women), aged between 18 and 88 years with BMI between 15·9 and 40·8 (kg/m2). The validation sample included 204 participants (110 women), aged between 18 and 86 years with BMI between 15·7 and 36·4 (kg/m2). Both samples included mixed ethnic/racial groups. All the participants underwent whole-body MRI to quantify TATM (dependent variable) and anthropometry (independent variables). Prediction equations developed using stepwise multiple regression were further investigated for agreement and bias before validation in separate data sets. Simplest equations with optimal R (2) and Bland-Altman plots demonstrated good agreement without bias in the validation analyses: men: TATM (kg)=0·198 weight (kg)+0·478 waist (cm)-0·147 height (cm)-12·8 (validation: R 2 0·79, CV=20 %, standard error of the estimate (SEE)=3·8 kg) and women: TATM (kg)=0·789 weight (kg)+0·0786 age (years)-0·342 height (cm)+24·5 (validation: R (2) 0·84, CV=13 %, SEE=3·0 kg). Published anthropometric prediction equations, based on MRI and computed tomographic scans, correlated strongly with MRI-measured TATM: (R (2) 0·70-0·82). Estimated TATFM correlated well with published prediction equations for total body fat based on underwater weighing (R (2) 0·70-0·80), with mean bias of 2·5-4·9 kg, correctable with log-transformation in most equations. In conclusion, new equations, using simple anthropometric measurements, estimated MRI-measured TATM with correlations and

  11. Quantification of three steroid hormone receptors of the leopard gecko (Eublepharis macularius), a lizard with temperature-dependent sex determination: their tissue distributions and the effect of environmental change on their expressions.

    PubMed

    Endo, Daisuke; Park, Min Kyun

    2003-12-01

    Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones function through their specific receptors, so the expression levels of the receptors may reflect the responsibility of target organs. However, there was no effective method to quantify the expression levels of these receptors in reptilian species. In this study, we established the competitive-PCR assay systems for the quantification of the mRNA expression levels of three sex steroid hormone receptors in the leopard gecko. These assay systems were successfully able to detect the mRNA expression level of each receptor in various organs of male adult leopard geckoes. The expression levels of mRNA of these receptors were highly various depending on the organs assayed. This is the first report regarding the tissue distributions of sex steroid hormone receptor expressions in reptile. The effects of environmental conditions on these hormone receptor expressions were also examined. After the low temperature and short photoperiod treatment for 6 weeks, only the androgen receptor expression was significantly increased in the testes. The competitive-PCR assay systems established in this report should be applicable for various studies of the molecular mechanism underlying the reproductive activity of the leopard gecko.

  12. Quantification of Paraquat, MPTP, and MPP+ in brain tissue using microwave-assisted solvent extraction (MASE) and high performance liquid chromatography-mass spectrometry

    PubMed Central

    Winnik, Bozena; Barr, Dana B.; Thiruchelvam, Mona; Montesano, M. Angela; Richfield, Eric K.; Buckley, Brian

    2014-01-01

    Animal models, consistent with the hypothesis of direct interaction of paraquat (PQ) and 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with specific areas of the central nervous system have been developed to study Parkinson’s disease (PD) in mice. These models have necessitated the creation of an analytical method for unambiguous identification and quantitation of PQ and structurally similar MPTP and 1-Methyl-4-phenylpyridinium ion (MPP+) in brain tissue. A method for determination of these compounds was developed using microwave-assisted solvent extraction (MASE) and liquid chromatography-mass spectrometry. Extraction solvent and microwave conditions such as power and time were optimized to produce recoveries of 90% for PQ 78% for MPTP and 97% for its metabolite MPP+. The chromatographic separation was performed on a C8, column and detection was carried out using an ion trap as an analyzer with electrospray ionization. Mass spectrometer parameters such as heated capillary temperature, spray voltage, capillary voltage and others were also optimized for each analyte. Analysis was done in Selective Ion Monitoring (SIM) mode using 186 m/z for PQ, m/z 174 for MPTP, and m/z 170 for MPP+. The method detection limit for paraquat in matrix was 100 pg and 40 pg for MPTP and 20 pg MPP+. PMID:19618168

  13. Comparative study of 13 candidate genes applying multi-reference normalization to detect the expression of different fineness in skin tissues of wool sheep.

    PubMed

    Tian, Y Z; Usman, T; Tian, K C; Di, J; Huang, X X; Xu, X M; Tulafu, H; Wu, W W; Fu, X F; Bai, Y; Tuerxun, M; Maimaiti, A

    2017-02-16

    Fiber diameter is a useful indicator of wool traits and it is the main determinant of wool quality and value. A comparative study was conducted to analyze the abundance and expression of 13 candidate genes using expression profile microarray analysis and to identify novel molecular markers associated with wool traits to provide a molecular basis for improving wool quality in sheep. Genes associated with fineness of skin tissue were identified using a real-time reverse transcriptase-polymerase chain reaction method with 18SrRNA, β-Actin, and GAPDH used for multi-reference normalization. The results indicated that the expression levels of TXNIP, TFDP1, and FAIM genes in super-fine type wool sheep were higher than those in fine-type wool sheep; the corresponding expression ratios of super-fine to fine wool sheep were 1.45, 1.57, and 2.55, respectively. The expression levels of PIK3CA, ADAM9, and FZD3 genes were lower in super-fine wool sheep compared with fine-type wool sheep; the corresponding expression ratios were 0.61, 0.65, and 0.52, respectively. The other genes tested (RPS6KA, ABCG2, GSTA1, PTPN13, GJB3, PPARD, and LAMB1) were similarly expressed in both types of wool sheep. These results infer that lower expression of PIK3CA, ADAM9, and FZD3 genes was associated with lower fiber diameter, whereas lower expression of TXNIP, TFDP1, and FAIM genes was associated with higher fiber diameter.

  14. Reference characterisation of sound speed and attenuation of the IEC agar-based tissue-mimicking material up to a frequency of 60 MHz.

    PubMed

    Rajagopal, Srinath; Sadhoo, Neelaksh; Zeqiri, Bajram

    2015-01-01

    To support the development of clinical applications of high-frequency ultrasound, appropriate tissue-mimicking materials (TMMs) are required whose acoustic properties have been measured using validated techniques. This paper describes the characterisation of the sound speed (phase velocity) and attenuation coefficient of the International Electrotechnical Commission (IEC) agar-based TMM over the frequency range 1 to 60 MHz. Measurements implemented a broadband through-transmission substitution immersion technique over two overlapping frequency ranges, with co-axially aligned 50 MHz centre-frequency transducers employed for characterisation above 15 MHz. In keeping with usual practice employed within the technical literature, thin acoustic windows (membranes) made of 12-μm-thick Mylar protected the TMM from water damage. Various important sources of uncertainty that could compromise measurement accuracy have been identified and evaluated through a combination of experimental studies and modelling. These include TMM sample thickness, measured both manually and acoustically, and the influence of interfacial losses that, even for thin protective membranes, are significant at the frequencies of interest. In agreement with previous reports, the attenuation coefficient of the IEC TMM exhibited non-linear frequency dependence, particularly above 20 MHz, yielding a value of 0.93 ± 0.04 dB cm(-1) MHz(-1) at 60 MHz, derived at 21 ± 0.5°C. For the first time, phase velocity, measured with an estimated uncertainty of ±3.1 m s(-1), has been found to be dispersive over this extended frequency range, increasing from 1541 m s(-1) at 1 MHz to 1547 m s(-1) at 60 MHz. This work will help standardise acoustic property measurements, and establishes a reference measurement capability for TMMs underpinning clinical applications at elevated frequencies.

  15. Identification and quantification of five macrolide antibiotics in several tissues, eggs and milk by liquid chromatography-electrospray tandem mass spectrometry.

    PubMed

    Dubois, M; Fluchard, D; Sior, E; Delahaut, P

    2001-04-05

    We present an electrospray high-performance liquid chromatographic tandem mass spectrometric (HPLC-MS-MS) method capable of determining in several tissues (muscle, kidney, liver), eggs and milk the following five macrolides: tylosin, tilmicosin, spiramycin, josamycin, erythromycin. Roxithromycin was used as an internal standard. The method uses extraction in a Tris buffer at pH 10.5, followed by protein precipitation with sodium tungstate and clean-up on an Oasis solid-phase extraction column. The HPLC separation was performed on a Purospher C18 column (125 x 3 mm I.D.) protected by a guard column, with a gradient of aqueous 0.1 M ammonium acetate-acetonitrile as the mobile phase at a flow-rate of 0.7 ml min(-1). Protonated molecules served as precursor ions for electrospray ionisation in the positive ion mode and four product ions were chosen for each analyte for multiple reaction monitoring (MRM). A validation study was conducted to confirm the five macrolides by MRM HPLC-MS-MS analysis of a negative control and fortified samples. All of the samples analysed were confirmed with four ions. The ion ratio reproducibility limit ranged from 2.4 to 15%. All compounds could be detected and quantified at half-maximum residue limits (MRLs). The method is specific, quantitative and reproducible enough to conform to European Union recommendations within the concentration range 0.5 MRL-2 MRL (accuracy: 80 to 110%, relative standard deviation: 2 to 13%). This whole method allows extraction and analysis of up to 50 samples per day.

  16. Confocal reference free traction force microscopy

    PubMed Central

    Bergert, Martin; Lendenmann, Tobias; Zündel, Manuel; Ehret, Alexander E.; Panozzo, Daniele; Richner, Patrizia; Kim, David K.; Kress, Stephan J. P.; Norris, David J.; Sorkine-Hornung, Olga; Mazza, Edoardo; Poulikakos, Dimos; Ferrari, Aldo

    2016-01-01

    The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology. Traction force microscopy (TFM) enables determination of the actuating forces. Despite progress, important limitations with intrusion effects in low resolution 2D pillar-based methods or disruptive intermediate steps of cell removal and substrate relaxation in high-resolution continuum TFM methods need to be overcome. Here we introduce a novel method allowing a one-shot (live) acquisition of continuous in- and out-of-plane traction fields with high sensitivity. The method is based on electrohydrodynamic nanodrip-printing of quantum dots into confocal monocrystalline arrays, rendering individually identifiable point light sources on compliant substrates. We demonstrate the undisrupted reference-free acquisition and quantification of high-resolution continuous force fields, and the simultaneous capability of this method to correlatively overlap traction forces with spatial localization of proteins revealed using immunofluorescence methods. PMID:27681958

  17. Identification and validation of reference genes for transcript normalization in strawberry (Fragaria × ananassa) defense responses.

    PubMed

    Amil-Ruiz, Francisco; Garrido-Gala, José; Blanco-Portales, Rosario; Folta, Kevin M; Muñoz-Blanco, Juan; Caballero, José L

    2013-01-01

    Strawberry (Fragaria spp) is an emerging model for the development of basic genomics and recombinant DNA studies among rosaceous crops. Functional genomic and molecular studies involve relative quantification of gene expression under experimental conditions of interest. Accuracy and reliability are dependent upon the choice of an optimal reference control transcript. There is no information available on validated endogenous reference genes for use in studies testing strawberry-pathogen interactions. Thirteen potential pre-selected strawberry reference genes were tested against different tissues, strawberry cultivars, biotic stresses, ripening and senescent conditions, and SA/JA treatments. Evaluation of reference candidate's suitability was analyzed by five different methodologies, and information was merged to identify best reference transcripts. A combination of all five methods was used for selective classification of reference genes. The resulting superior reference genes, FaRIB413, FaACTIN, FaEF1α and FaGAPDH2 are strongly recommended as control genes for relative quantification of gene expression in strawberry. This report constitutes the first systematic study to identify and validate optimal reference genes for accurate normalization of gene expression in strawberry plant defense response studies.

  18. Identification and Validation of Reference Genes for Transcript Normalization in Strawberry (Fragaria × ananassa) Defense Responses

    PubMed Central

    Amil-Ruiz, Francisco; Garrido-Gala, José; Blanco-Portales, Rosario; Folta, Kevin M.; Muñoz-Blanco, Juan; Caballero, José L.

    2013-01-01

    Strawberry (Fragaria spp) is an emerging model for the development of basic genomics and recombinant DNA studies among rosaceous crops. Functional genomic and molecular studies involve relative quantification of gene expression under experimental conditions of interest. Accuracy and reliability are dependent upon the choice of an optimal reference control transcript. There is no information available on validated endogenous reference genes for use in studies testing strawberry-pathogen interactions. Thirteen potential pre-selected strawberry reference genes were tested against different tissues, strawberry cultivars, biotic stresses, ripening and senescent conditions, and SA/JA treatments. Evaluation of reference candidate’s suitability was analyzed by five different methodologies, and information was merged to identify best reference transcripts. A combination of all five methods was used for selective classification of reference genes. The resulting superior reference genes, FaRIB413, FaACTIN, FaEF1α and FaGAPDH2 are strongly recommended as control genes for relative quantification of gene expression in strawberry. This report constitutes the first systematic study to identify and validate optimal reference genes for accurate normalization of gene expression in strawberry plant defense response studies. PMID:23940602

  19. Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real-time PCR.

    PubMed

    Batista, Ribrio I T P; Luciano, Maria C S; Teixeira, Dárcio I A; Freitas, Vicente J F; Melo, Luciana M; Andreeva, Lyudmila E; Serova, Irina A; Serov, Oleg L

    2014-01-01

    Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte-colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra-assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild-type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin-based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue-specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.

  20. Plasma membrane calcium ATPase (PMCA4): A housekeeper for RT-PCR relative quantification of polytopic membrane proteins

    PubMed Central

    Calcagno, Anna Maria; Chewning, Katherine J; Wu, Chung-Pu; Ambudkar, Suresh V

    2006-01-01

    Background Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters. Results In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line. Conclusion We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors. PMID:16978418

  1. Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in the Half-Smooth Tongue Sole (Cynoglossus semilaevis) at Different Developmental Stages, in Various Tissue Types and on Exposure to Chemicals

    PubMed Central

    Liu, Conghui; Xin, Nian; Zhai, Yi; Jiang, Liming; Zhai, Jieming; Zhang, Quanqi; Qi, Jie

    2014-01-01

    Quantitative real time RT-PCR has been described as the most sensitive method for the detection of low abundance mRNA. To date, no reference genes have been screened in the half-smooth tongue sole (Cynoglossus semilaevis). The aim of this study was to select the most stable genes for quantitative real-time RT-PCR. Eight housekeeping genes (18S, TUBA, B2M, ACTB, EF1A, GAPDH, RPL17 and UBCE) were tested at different developmental stages, in different tissues, and following exposure to the drug SB-431542. Using geNorm, BestKeeper and NormFinder software, GAPDH/B2M, GAPDH/18S and UBCE/GAPDH were identified as the most suitable genes from samples taken of different developmental stages while 18S/RPL17 were consistently ranked as the best reference genes for different tissue types. Furthermore, TUBA/B2M, TUBA/UBCE and B2M/TUBA were found to be the most suitable genes in samples treated with the drug, SB-431542 by geNorm, BestKeeper and NormFinder respectively. Across both different developmental stages and tissue types, the combination of 18S and GAPDH was the most stable reference gene analyzed by Ref-Finder. To test and verify the screened reference genes, the expression profiles of LEFTY-normalized to the combination of GAPDH/18S and ACTB were presented. These results will be useful for future gene-expression studies in the half-smooth tongue sole. PMID:24667563

  2. References for marine science

    NASA Astrophysics Data System (ADS)

    1990-06-01

    Standard and Reference Materials for Marine Science, National Oceanic and Atmospheric Administration Technical Memo OMA-51 (2nd edition, 434 pp.), by A. Y. Cantillo, is now available. This compilation of reference materials was prepared at the request of the Group of Experts on Standards and Reference Materials and was printed by NOAA. GESREM is sponsored by the International Atomic Energy Agency, the Intergovernmental Oceanographic Commission, and the United Nations Program.Reference materials are included on ashes, gases, instrument performance materials, oils, physical properties, rocks, sediments, sludges, tissues and waters. For each reference material, source, description and preparation, analyses and values, cost, references, and comments are given. Indices are included for elements, isotopes and organic compounds. Cross references to Chemical Abstracts Service registry numbers and alternate names and chemical structures of organic compounds are also provided.

  3. Optically transmitted and inductively coupled electric reference to access in vivo concentrations for quantitative proton-decoupled ¹³C magnetic resonance spectroscopy.

    PubMed

    Chen, Xing; Pavan, Matteo; Heinzer-Schweizer, Susanne; Boesiger, Peter; Henning, Anke

    2012-01-01

    This report describes our efforts on quantification of tissue metabolite concentrations in mM by nuclear Overhauser enhanced and proton decoupled (13) C magnetic resonance spectroscopy and the Electric Reference To access In vivo Concentrations (ERETIC) method. Previous work showed that a calibrated synthetic magnetic resonance spectroscopy-like signal transmitted through an optical fiber and inductively coupled into a transmit/receive coil represents a reliable reference standard for in vivo (1) H magnetic resonance spectroscopy quantification on a clinical platform. In this work, we introduce a related implementation that enables simultaneous proton decoupling and ERETIC-based metabolite quantification and hence extends the applicability of the ERETIC method to nuclear Overhauser enhanced and proton decoupled in vivo (13) C magnetic resonance spectroscopy. In addition, ERETIC signal stability under the influence of simultaneous proton decoupling is investigated. The proposed quantification method was cross-validated against internal and external reference standards on human skeletal muscle. The ERETIC signal intensity stability was 100.65 ± 4.18% over 3 months including measurements with and without proton decoupling. Glycogen and unsaturated fatty acid concentrations measured with the ERETIC method were in excellent agreement with internal creatine and external phantom reference methods, showing a difference of 1.85 ± 1.21% for glycogen and 1.84 ± 1.00% for unsaturated fatty acid between ERETIC and creatine-based quantification, whereas the deviations between external reference and creatine-based quantification are 6.95 ± 9.52% and 3.19 ± 2.60%, respectively.

  4. Stereology and morphometry of lung tissue.

    PubMed

    Mühlfeld, Christian; Knudsen, Lars; Ochs, Matthias

    2013-01-01

    This chapter deals with the stereological quantification of structural characteristics of the lung. The aim of design-based stereological methods is the unbiased and efficient estimation of structural features without making any assumptions on the underlying nature of the biological sample. The methods are based on rigorous sampling of location and orientation, the application of appropriate test systems, and the controlling of the precision of the estimates. Here, we describe the workflow from the fixation of the lung over the processing of the tissue samples to gaining estimates on the structural properties of the lung. Specifically, this chapter deals with methods for estimating the reference volume, sampling location, and sampling orientation, estimating volumes and surface areas of alveolar compartments, estimating total alveolar number, performing stereology at light and electron microscopic level, and dealing with technical problems such as tissue shrinkage. The procedures are illustrated using a worked example from the authors' own laboratory.

  5. Quantification of dopamine transporter density with [18F]FECNT PET in healthy humans

    PubMed Central

    Nye, Jonathon A.; Votaw, John R.; Bremner, J. Douglas; Davis, Margaret R.; Voll, Ronald J.; Camp, Vernon M.; Goodman, Mark M.

    2015-01-01

    Introduction Fluorine-18 labeled 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane ([18 F]FECNT) binds reversibly to the dopamine transporter (DAT) with high selectivity. [18 F]FECNT has been used extensively in the quantification of DAT occupancy in non-human primate brain and can distinguish between Parkinson's and healthy controls in humans. The purpose of this work was to develop a compartment model to characterize the kinetics of [18 F]FECNT for quantification of DAT density in healthy human brain. Methods Twelve healthy volunteers underwent 180 min dynamic [18 F]FECNT PET imaging including sampling of arterial blood. Regional time-activity curves were extracted from the caudate, putamen and midbrain including a reference region placed in the cerebellum. Binding potential, BPND, was calculated for all regions using kinetic parameters estimated from compartmental and Logan graphical model fits to the time-activity data. Simulations were performed to determine whether the compartment model could reliably fit time-activity data over a range of BPND values. Results The kinetics of [18 F]FECNT were well-described by the reversible 2-tissue arterial input and full reference tissue compartment models. Calculated binding potentials in the caudate, putamen and midbrain were in good agreement between the arterial input model, reference tissue model and the Logan graphical model. The distribution volume in the cerebellum did not reach a plateau over the duration of the study, which may be a result of non-specific binding in the cerebellum. Simulations that included non-specific binding show that the reference and arterial input models are able to estimate BPND for DAT densities well below that observed in normal volunteers. Conclusion The kinetics of [18 F]FECNT in human brain are well-described by arterial input and reference tissue compartment models. Measured and simulated data show that BPND calculated with reference tissue model is proportional to

  6. Tissue distribution and subcellular localization of trace metals in the pond snail Lymnaea stagnalis with special reference to the role of lysosomal granules in metal sequestration.

    PubMed

    Desouky, Mahmoud M A

    2006-05-01

    The present study was undertaken to elucidate the cellular mechanisms, which govern metal sequestration and detoxification in gastropods. For this purpose the pond snail, Lymnaea stagnalis was exposed to environmentally relevant concentrations of three species of metals (Al, Zn and Cd) for 30 days and the localization and fate of these metals were followed in different tissues of the snails. The measurement of relative distribution of metals between tissues revealed that the digestive gland and kidney account for most of the accumulated metals. Al and Cd (non-essential metals) were redistributed to the digestive gland, possibly because of the presence of specific binding entities in the digestive glands of the herein species. This study focuses on the role of intracellular metal-containing granules on metal sequestration. Three main types of granules were identified in the digestive gland cells namely small, green and yellow granules. The morphological examination and the progressive accumulation of elements within these granules revealed that they are developmental stages with the yellow granule being the mature one. The total number of these granules was found to be significantly increased upon exposure of the snails to Al only. This increase may be a response to the large amount of Al that is accumulated through feeding route of this grazing snail. X-ray microanalysis (XRMA) revealed that metals were localized in all three types of digestive gland granules. The increased amount of ligands (P and S) in the granules may give evidence for their role in metal sequestration. Levels of Al and P were positively correlated in the digestive gland granules. It is possible that aluminium is bound to phosphorus to render it insoluble and so to both immobilize it within the lysosome and to be excreted in a highly insoluble form. On the other hand, both Zn and Cd induced marked upregulation of S in mature (yellow) granules by 26- and 11-folds, respectively. The lysosomal

  7. Optical probe with reference fiber

    DOEpatents

    Da Silva, Luiz B.; Chase, Charles L.

    2006-03-14

    A system for characterizing tissue includes the steps of generating an emission signal, generating a reference signal, directing the emission signal to and from the tissue, directing the reference signal in a predetermined manner relative to the emission signal, and using the reference signal to compensate the emission signal. In one embodiment compensation is provided for fluctuations in light delivery to the tip of the probe due to cable motion.

  8. Selection and Validation of Reference Genes for qRT-PCR in Cycas elongata

    PubMed Central

    Deng, Tian; Chen, Letian; Wu, Hong; Zhang, Shouzhou

    2016-01-01

    Quantitative reverse transcription PCR (qRT-PCR) is a sensitive technique used in gene expression studies. To achieve a reliable quantification of transcripts, appropriate reference genes are required for comparison of transcripts in different samples. However, few reference genes are available for non-model taxa, and to date, reliable reference genes in Cycas elongata have not been well characterized. In this study, 13 reference genes (ACT7, TUB, UBQ, EIF4, EF1, CLATHRIN1, PP2A, RPB2, GAPC2, TIP41, MAPK, SAMDC and CYP) were chosen from the transcriptome database of C. elongata, and these genes were evaluated in 8 different organ samples. Three software programs, NormFinder, GeNorm and BestKeeper, were used to validate the stability of the potential reference genes. Results obtained from these three programs suggested that CeGAPC2 and CeRPB2 are the most stable reference genes, while CeACT7 is the least stable one among the 13 tested genes. Further confirmation of the identified reference genes was established by the relative expression of AGAMOUSE gene of C. elongata (CeAG). While our stable reference genes generated consistent expression patterns in eight tissues, we note that our results indicate that an inappropriate reference gene might cause erroneous results. Our systematic analysis for stable reference genes of C. elongata facilitates further gene expression studies and functional analyses of this species. PMID:27124298

  9. Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

    PubMed Central

    Ling, Hui; Wu, Qibin; Guo, Jinlong; Xu, Liping; Que, Youxiong

    2014-01-01

    The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species. PMID:24823940

  10. Evaluation of Reference Genes for Quantitative Real-Time PCR in Songbirds

    PubMed Central

    Zinzow-Kramer, Wendy M.; Horton, Brent M.; Maney, Donna L.

    2014-01-01

    Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbird: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology. PMID:24780145

  11. Reference Services.

    ERIC Educational Resources Information Center

    Bunge, Charles A.

    1999-01-01

    Discusses library reference services. Topics include the historical development of reference services; instruction in library use, particularly in college and university libraries; guidance; information and referral services and how they differ from traditional question-answering service; and future concerns, including user fees and the planning…

  12. Reference Assessment

    ERIC Educational Resources Information Center

    Bivens-Tatum, Wayne

    2006-01-01

    This article presents interesting articles that explore several different areas of reference assessment, including practical case studies and theoretical articles that address a range of issues such as librarian behavior, patron satisfaction, virtual reference, or evaluation design. They include: (1) "Evaluating the Quality of a Chat Service"…

  13. Reference Revolutions.

    ERIC Educational Resources Information Center

    Mason, Marilyn Gell

    1998-01-01

    Describes developments in Online Computer Library Center (OCLC) electronic reference services. Presents a background on networked cataloging and the initial implementation of reference services by OCLC. Discusses the introduction of OCLC FirstSearch service, which today offers access to over 65 databases, future developments in integrated…

  14. Quantification of γ-aminobutyric acid (GABA) in (1) H MRS volumes composed heterogeneously of grey and white matter.

    PubMed

    Mikkelsen, Mark; Singh, Krish D; Brealy, Jennifer A; Linden, David E J; Evans, C John

    2016-11-01

    The quantification of γ-aminobutyric acid (GABA) concentration using localised MRS suffers from partial volume effects related to differences in the intrinsic concentration of GABA in grey (GM) and white (WM) matter. These differences can be represented as a ratio between intrinsic GABA in GM and WM: rM . Individual differences in GM tissue volume can therefore potentially drive apparent concentration differences. Here, a quantification method that corrects for these effects is formulated and empirically validated. Quantification using tissue water as an internal concentration reference has been described previously. Partial volume effects attributed to rM can be accounted for by incorporating into this established method an additional multiplicative correction factor based on measured or literature values of rM weighted by the proportion of GM and WM within tissue-segmented MRS volumes. Simulations were performed to test the sensitivity of this correction using different assumptions of rM taken from previous studies. The tissue correction method was then validated by applying it to an independent dataset of in vivo GABA measurements using an empirically measured value of rM . It was shown that incorrect assumptions of rM can lead to overcorrection and inflation of GABA concentration measurements quantified in volumes composed predominantly of WM. For the independent dataset, GABA concentration was linearly related to GM tissue volume when only the water signal was corrected for partial volume effects. Performing a full correction that additionally accounts for partial volume effects ascribed to rM successfully removed this dependence. With an appropriate assumption of the ratio of intrinsic GABA concentration in GM and WM, GABA measurements can be corrected for partial volume effects, potentially leading to a reduction in between-participant variance, increased power in statistical tests and better discriminability of true effects.

  15. Importance of reference gene selection for articular cartilage mechanobiology studies

    PubMed Central

    Al-Sabah, A.; Stadnik, P.; Gilbert, S.J.; Duance, V.C.; Blain, E.J.

    2016-01-01

    Summary Objective Identification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively. Design The expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder. Results Appropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions. Conclusion This study demonstrates that commonly used ‘reference genes’ may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to

  16. Colour thresholding and objective quantification in bioimaging

    NASA Technical Reports Server (NTRS)

    Fermin, C. D.; Gerber, M. A.; Torre-Bueno, J. R.

    1992-01-01

    Computer imaging is rapidly becoming an indispensable tool for the quantification of variables in research and medicine. Whilst its use in medicine has largely been limited to qualitative observations, imaging in applied basic sciences, medical research and biotechnology demands objective quantification of the variables in question. In black and white densitometry (0-256 levels of intensity) the separation of subtle differences between closely related hues from stains is sometimes very difficult. True-colour and real-time video microscopy analysis offer choices not previously available with monochrome systems. In this paper we demonstrate the usefulness of colour thresholding, which has so far proven indispensable for proper objective quantification of the products of histochemical reactions and/or subtle differences in tissue and cells. In addition, we provide interested, but untrained readers with basic information that may assist decisions regarding the most suitable set-up for a project under consideration. Data from projects in progress at Tulane are shown to illustrate the advantage of colour thresholding over monochrome densitometry and for objective quantification of subtle colour differences between experimental and control samples.

  17. Ready Reference.

    ERIC Educational Resources Information Center

    Koltay, Emery

    1999-01-01

    Includes the following ready reference information: "Publishers' Toll-Free Telephone Numbers"; "How to Obtain an ISBN (International Standard Book Number)"; "How to Obtain an ISSN (International Standard Serial Number)"; and "How to Obtain an SAN (Standard Address Number)". (AEF)

  18. MicroRNAs are suitable for assessment as biomarkers from formalin-fixed paraffin-embedded tissue, and miR-24 represents an appropriate reference microRNA for diffuse large B-cell lymphoma studies.

    PubMed

    Culpin, Rachel Emily; Sieniawski, Michal; Proctor, Stephen John; Menon, Geetha; Mainou-Fowler, Tryfonia

    2013-03-01

    Tissue biopsy specimens in the form of formalin-fixed paraffin-embedded tissue (FFPET) represent a valuable resource for biomarker identification and validation. However, to date, they remain an underused asset due to uncertainty regarding RNA extraction and the reliability of downstream techniques, including quantitative RT-PCR. Recently, much interest has emerged in the study of microRNAs; small single-stranded RNAs with a role in transcriptional regulation, that are thought to be well preserved in FFPET. In this study, we show that microRNA expression is comparable between FFPET and matched fresh-frozen samples (miR-17-5p: p=0.01, miR-92: p=0.003), and demonstrate that no significant deterioration in expression occurs over prolonged FFPET storage (p=0.06). Furthermore, microRNA expression is equivalent dependant on RNA extraction method (p<0.001) or DNAse treatment of total RNA (p<0.001). Finally, we validate miR-24 as a suitable reference microRNA for diffuse large B-cell lymphoma (DLBCL) FFPET studies.

  19. Identification of suitable reference genes for investigating gene expression in human gallbladder carcinoma using reverse transcription quantitative polymerase chain reaction.

    PubMed

    Yu, Shan; Yang, Qiwei; Yang, Jing Hui; Du, Zhenwu; Zhang, Guizhen

    2015-04-01

    Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT‑qPCR data. The key aim of this method is to identify an applicable internal reference gene, however, there are currently no suitable reference genes for gene analysis in gallbladder carcinoma. In the present study, screening was performed using 12 common reference genes, which were selected in order to provide an experimental basis for the investigation of gene expression in gallbladder carcinoma. A total of 16 tissue samples of gallbladder carcinoma and their matched normal gallbladder tissues were used. The gene expression stability and applicability of the 12 reference gene candidates were determined using the geNorm, NormFinder and BestKeeper software programs. Following comparison of the results of the three software programs, HPRT1 was identified as the most stably expressed reference gene. In the normal gallbladder group, the relative stably expressed reference gene was PPIA and in the entire sample group, the relatively stably expressed reference gene was PPIA. The present study also demonstrated that the combination of the three reference genes was the most appropriate. The recommended combinations were PPIA + PUM1 + ACTB for the total sample group, GAPDH + PBGD + ALAS1 for the gallbladder carcinoma group and PPIA + PUM1 + TBP for the paired normal gallbladder group.

  20. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-06

    Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.

  1. Recent application of quantification II in Japanese medical research.

    PubMed Central

    Suzuki, T; Kudo, A

    1979-01-01

    Hayashi's Quantification II is a method of multivariate discrimination analysis to manipulate attribute data as predictor variables. It is very useful in the medical research field for estimation, diagnosis, prognosis, evaluation of epidemiological factors, and other problems based on multiplicity of attribute data. In Japan, this method is so well known that most of the computer program packages include the Hayashi Quantification, but it seems to be yet unfamiliar with the method for researchers outside Japan. In view of this situation, we introduced 19 selected articles of recent applications of the Quantification II in Japanese medical research. In reviewing these papers, special mention is made to clarify how the researchers were satisfied with findings provided by the method. At the same time, some recommendations are made about terminology and program packages. Also a brief discussion of the background of the quantification methods is given with special reference to the Behaviormetric Society of Japan. PMID:540587

  2. Validation of Suitable Reference Genes for Assessing Gene Expression of MicroRNAs in Lonicera japonica

    PubMed Central

    Wang, Yaolong; Liu, Juan; Wang, Xumin; Liu, Shuang; Wang, Guoliang; Zhou, Junhui; Yuan, Yuan; Chen, Tiying; Jiang, Chao; Zha, Liangping; Huang, Luqi

    2016-01-01

    MicroRNAs (miRNAs), which play crucial regulatory roles in plant secondary metabolism and responses to the environment, could be developed as promising biomarkers for different varieties and production areas of herbal medicines. However, limited information is available for miRNAs from Lonicera japonica, which is widely used in East Asian countries owing to various pharmaceutically active secondary metabolites. Selection of suitable reference genes for quantification of target miRNA expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of secondary metabolic regulation in different tissues and varieties of L. japonica. For precise normalization of gene expression data in L. japonica, 16 candidate miRNAs were examined in three tissues, as well as 21 cultivated varieties collected from 16 production areas, using GeNorm, NormFinder, and RefFinder algorithms. Our results revealed combination of u534122 and u3868172 as the best reference genes across all samples. Their specificity was confirmed by detecting the cycling threshold (Ct) value ranges in different varieties of L. japonica collected from diverse production areas, suggesting the use of these two reference miRNAs is sufficient for accurate transcript normalization with different tissues, varieties, and production areas. To our knowledge, this is the first report on validation of reference miRNAs in honeysuckle (Lonicera spp.). Restuls from this study can further facilitate discovery of functional regulatory miRNAs in different varieties of L. japonica. PMID:27507983

  3. Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species.

    PubMed

    Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

    2016-01-01

    Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

  4. Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

    PubMed Central

    Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

    2016-01-01

    Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses. PMID:26863232

  5. Poroelastic references

    SciTech Connect

    Morency, Christina

    2014-12-12

    This file contains a list of relevant references on the Biot theory (forward and inverse approaches), the double-porosity and dual-permeability theory, and seismic wave propagation in fracture porous media, in RIS format, to approach seismic monitoring in a complex fractured porous medium such as Brady?s Geothermal Field.

  6. Ready Reference.

    ERIC Educational Resources Information Center

    Koltay, Emery

    2001-01-01

    Includes four articles that relate to ready reference, including a list of publishers' toll-free telephone numbers and Web sites; how to obtain an ISBN (International Standard Book Number) and an ISSN (International Standard Serial Number); and how to obtain an SAN (Standard Address Number), for organizations that are involved in the book…

  7. Quantification Of Tissue Effects Due To A Pulsed Er:Yag Laser At 2.9 μm With Beam Delivery In A Wet Field Via Zirconium Fluoride Fibers

    NASA Astrophysics Data System (ADS)

    Bonner, R. F.; Smith, P. D.; Leon, M.; Esterowitz, L.; Storm, M.; Levin, K.; Tran, D.

    1987-04-01

    Prototype Er:YAG lasers were used to generate 70-170 usec long pulses of 2.9 urn containing a series of lusec relaxation oscillation spikes which were coupled into prototype zirconium fluoride glass optical fibers of 180-250 urn diameter. Pulse energy densities in excess of 2400 mJ/mm2 were transmitted through these fibers without damage. Systematic studies of tissue damage by the Er:YAG laser pulses delivered to the surface of the tissue in a wet field via the ZrF4 fibers demonstrated effective ablative thresholds of 5 mJ/mm2 and slope ablative efficiency of 0.5 mm3/J for soft tissue such as human cadaver coronary artery. For heavily calcified tissues such as hard human calcified atherosclerotic plaque or rib bone ablative thresholds were increased 3 fold and the slope efficiency was slighlty decreased. Tissue thermal damage zone was confined to a region of 3-5 urn from the edge of the ablated zone for suprathreshold pulses. These studies demonstrate the feasibility of an Er:YAG laser - zirconium fluoride fiber microsurgical system for laser angioplasty and other forms of remote human ablative microsurgery.

  8. Reference Man anatomical model

    SciTech Connect

    Cristy, M.

    1994-10-01

    The 70-kg Standard Man or Reference Man has been used in physiological models since at least the 1920s to represent adult males. It came into use in radiation protection in the late 1940s and was developed extensively during the 1950s and used by the International Commission on Radiological Protection (ICRP) in its Publication 2 in 1959. The current Reference Man for Purposes of Radiation Protection is a monumental book published in 1975 by the ICRP as ICRP Publication 23. It has a wealth of information useful for radiation dosimetry, including anatomical and physiological data, gross and elemental composition of the body and organs and tissues of the body. The anatomical data includes specified reference values for an adult male and an adult female. Other reference values are primarily for the adult male. The anatomical data include much data on fetuses and children, although reference values are not established. There is an ICRP task group currently working on revising selected parts of the Reference Man document.

  9. Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses

    PubMed Central

    Zhang, Yunxing; Han, Xiaojiao; Chen, Shuangshuang; Zheng, Liu; He, Xuelian; Liu, Mingying; Qiao, Guirong; Wang, Yang; Zhuo, Renying

    2017-01-01

    Salix matsudana is a deciduous, rapidly growing willow species commonly cultivated in China, which can tolerate drought, salt, and heavy metal stress conditions. Selection of suitable reference genes for quantitative real-time PCR is important for normalizing the expression of the key genes associated with various stresses. To validate suitable reference genes, we selected 11 candidate reference genes (five traditional housekeeping genes and six novel genes) and analyzed their expression stability in various samples, including different tissues and under different abiotic stress treatments. The expression of these genes was determined using five programs—geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder. The results showed that α-TUB2 (alpha-tubulin 2) and DnaJ (chaperone protein DnaJ 49) were the most stable reference genes across all the tested samples. We measured the expression profiles of the defense response gene SmCAT (catalase) using the two most stable and one least stable reference genes in all samples of S. matsudana. The relative quantification of SmCAT varied greatly according to the different reference genes. We propose that α-TUB2 and DnaJ should be the preferred reference genes for normalization and quantification of transcript levels in future gene expression studies in willow species under various abiotic stress conditions. PMID:28120870

  10. Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius.

    PubMed

    Mamidala, Praveen; Rajarapu, Swapna P; Jones, Susan C; Mittapalli, Omprakash

    2011-07-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.

  11. Cranial Base Superimposition for 3D Evaluation of Soft Tissue Changes

    PubMed Central

    Cevidanes, Lucia H.C.; Motta, Alexandre; Proffit, William R.; Ackerman, James L.; Styner, Martin

    2009-01-01

    The recent emphasis on soft tissues as the limiting factor in treatment and on soft tissue relationships in establishing the goals of treatment has made 3D analysis of soft tissues more important in diagnosis and treatment planning. It is equally important to be able to detect changes in the facial soft tissues produced by growth and/or treatment. This requires structures of reference for superimposition, and a way to display the changes with quantitative information. This paper outlines a technique for quantifying facial soft tissue changes as viewed in CBCT data, using fully-automated voxel-wise registration of the cranial base surface. The assessment of change of soft tissues is done via calculation of the Euclidean surface distances between the 3D models. Color maps are used for visual assessment of the location and quantification of changes. This methodology allows a detailed examination of soft tissue changes with growth and/or treatment. Because of the lack of stable references with 3D photogrammetry, 3D photography and laser scanning, soft tissue changes cannot be accurately quantified by these methods. PMID:20381752

  12. Colorimetric Quantification and in Situ Detection of Collagen

    ERIC Educational Resources Information Center

    Esteban, Francisco J.; del Moral, Maria L.; Sanchez-Lopez, Ana M.; Blanco, Santos; Jimenez, Ana; Hernandez, Raquel; Pedrosa, Juan A.; Peinado, Maria A.

    2005-01-01

    A simple multidisciplinary and inexpensive laboratory exercise is proposed, in which the undergraduate student may correlate biochemical and anatomical findings. The entire practical session can be completed in one 2.5-3 hour laboratory period, and consists of the quantification of collagen and total protein content from tissue sections--without…

  13. Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut

    PubMed Central

    Cindhuri, Katamreddy Sri; Sharma, Kiran K.

    2013-01-01

    The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut. PMID:24167633

  14. Evaluation and validation of reference genes for normalization of quantitative real-time PCR based gene expression studies in peanut.

    PubMed

    Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Cindhuri, Katamreddy Sri; Sharma, Kiran K

    2013-01-01

    The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.

  15. NMR-based quantification of organic diphosphates

    PubMed Central

    Lenevich, Stepan

    2010-01-01

    Phosphorylated compounds are ubiquitous in life. Given their central role, many such substrates and analogues have been prepared for subsequent evaluation. Prior to biological experiments, it is typically necessary to determine the concentration of the target molecule in solution. Here we describe a method where concentrations of stock solutions of organic diphosphates and bisphosphonates are quantified using 31P NMR spectroscopy with standard instrumentation using a capillary tube with a secondary standard. The method is specific and is applicable down to a concentration of 200 μM. The capillary tube provides the reference peak for quantification and deuterated solvent for locking. PMID:20833124

  16. Detection and quantification of poly-ADP-ribosylated cellular proteins of spleen and liver tissues of mice in vivo by slot and Western blot immunoprobing using polyclonal antibody against mouse ADP-ribose polymer.

    PubMed

    Sharan, R N; Devi, B Jaylata; Humtsoe, J O; Saikia, Jyoti R; Kma, L

    2005-10-01

    Poly-ADP-ribosylation (PAR) of cellular proteins has been shown to have decisive roles in diverse cellular functions including carcinogenesis. There are indications that metabolic level of poly-ADP-ribosylated cellular proteins might indicate carcinogenesis and, therefore, could be potentially used in cancer screening program. Keeping in mind the limitations of currently available assays of cellular PAR, a new assay is being reported that measures the metabolic level of poly-ADP-ribosylated cellular proteins. The ELISA based slot and Western blot immunoassay used polyclonal antibody against natural, heterogeneous ADP-ribose polymers. It could be successfully employed to qualitatively and quantitatively assay metabolic levels of poly-ADP-ribosylated proteins of spleen and liver tissues of normal mice or mice exposed to dimethylnitrosamine for up to 8 weeks; potentially PAR of cellular proteins could be assayed in any tissue or biopsy. Implications of the results in cancer screening program have been discussed.

  17. Quantification and kinetic study in plasma and tissues of (E)-1,1,4,4-tetramethyl-2-tetrazene, a liquid propellant and a transformation product of 1,1-dimethyl hydrazine.

    PubMed

    Payen, Léa; Honorat, Mylène; Bouard, Charlotte; Jacob, Guy; Terreux, Raphael; Delalu, Henri; Labarthe, Emilie; Guitton, Jérôme

    2015-09-01

    (E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ) is formed from the oxidation of the unsymmetrical 1,1-dimethylhydrazine (UDMH) and is used as a storable liquid fuel which can be considered as a new potential propellant for space rocket propulsion. To better understand the toxicological behavior of the compound, an intraperitoneal administration of TMTZ was performed in mice to define its toxicokinetics and tissue distribution. A fully validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assay was developed to determine TMTZ levels in biological samples. Determination of TMTZ was achieved using 50 μL of plasma or tissue solution. Precipitation with ammonium sulfate and acetonitrile was used for sample preparation. Liquid chromatography was performed on an Atlantis HILIC Silica column (Waters; 3 μm, 150 mm × 2.1 mm i.d.). Isocratic elution with a mixture of ammonium acetate buffer (pH 5, 100 mM)/water/acetonitrile (3:2:95, v/v/v) was used. The detection was conducted using an electrospray source in positive ion mode. TMTZ and (15)N2-TMTZ (internal standard) were quantitated in selected reaction monitoring mode using the transition m/z 117→72 and 119→74, respectively. Standard curves exhibited excellent linearity in the range of 10-500 ng/mL for plasma and 50-2000 ng/mL for all tissues (heart, liver, brain, kidney, and lung) analyzed, and acceptable precision and accuracy (<10 %) were obtained. The elimination rate constant strongly suggests that TMTZ was very quickly eliminated from the body. The results of tissue distribution experiments indicated that TMTZ underwent a rapid distribution into limited organs such as the liver, kidney, and brain.

  18. mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome

    PubMed Central

    Puisac, Beatriz; Teresa-Rodrigo, María-Esperanza; Hernández-Marcos, María; Baquero-Montoya, Carolina; Gil-Rodríguez, María-Concepción; Visnes, Torkild; Bot, Christopher; Gómez-Puertas, Paulino; Kaiser, Frank J.; Ramos, Feliciano J.; Ström, Lena; Pié, Juan

    2017-01-01

    Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers. PMID:28241484

  19. mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome.

    PubMed

    Puisac, Beatriz; Teresa-Rodrigo, María-Esperanza; Hernández-Marcos, María; Baquero-Montoya, Carolina; Gil-Rodríguez, María-Concepción; Visnes, Torkild; Bot, Christopher; Gómez-Puertas, Paulino; Kaiser, Frank J; Ramos, Feliciano J; Ström, Lena; Pié, Juan

    2017-02-23

    Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.

  20. Modeling Strategies for Quantification of In Vivo (18)F-AV-1451 Binding in Patients with Tau Pathology.

    PubMed

    Hahn, Andreas; Schain, Martin; Erlandsson, Maria; Sjölin, Petter; James, Gregory M; Strandberg, Olof T; Hägerström, Douglas; Lanzenberger, Rupert; Jögi, Jonas; Olsson, Tomas G; Smith, Ruben; Hansson, Oskar

    2017-04-01

    Aggregation of hyperphosphorylated tau is a major hallmark of many neurodegenerative diseases, including Alzheimer disease (AD). In vivo imaging with PET may offer important insights into pathophysiologic mechanisms, diagnosis, and disease progression. We describe different strategies for quantification of (18)F-AV-1451 (T807) tau binding, including models with blood sampling and noninvasive alternatives. Methods: Fifteen subjects (4 controls, 6 AD, 3 progressive supranuclear palsy, 2 cortico basal syndrome) underwent 180-min PET with (18)F-AV-1451 and arterial blood sampling. Modeling with arterial input functions included 1-, 2-, and 3-tissue-compartment models and the Logan plot. Using the cerebellum as reference region, we applied the simplified reference tissue model 2 and Logan reference plot. Finally, simplified outcome measures were calculated as ratio, with reference to cerebellar concentrations (SUV ratio [SUVR]) and SUVs. Results: Tissue compartment models were not able to describe the kinetics of (18)F-AV-1451, with poor fits in 33%-53% of cortical regions and 80% in subcortical areas. In contrast, the Logan plot showed excellent fits and parameter variance (total volume of distribution SE < 5%). Compared with the 180-min arterial-based Logan model, strong agreement was obtained for the Logan reference plot also for a reduced scan time of 100 min (R(2) = 0.91) and SUVR 100-120 min (R(2) = 0.94), with 80-100 min already representing a reasonable compromise between duration and accuracy (R(2) = 0.93). Time-activity curves and kinetic parameters were equal for cortical regions and the cerebellum in control subjects but different in the putamen. Cerebellar total volumes of distribution were higher in controls than patients. For these methods, increased cortical binding was observed for AD patients and to some extent for cortico basal syndrome, but not progressive supranuclear palsy. Conclusion: The Logan plot provided the best estimate of tau binding using

  1. Utilizing a reference material for assessing absolute tumor mechanical properties in modality independent elastography

    NASA Astrophysics Data System (ADS)

    Kim, Dong Kyu; Weis, Jared A.; Yankeelov, Thomas E.; Miga, Michael I.

    2014-03-01

    There is currently no reliable method for early characterization of breast cancer response to neoadjuvant chemotherapy (NAC) [1,2]. Given that disruption of normal structural architecture occurs in cancer-bearing tissue, we hypothesize that further structural changes occur in response to NAC. Consequently, we are investigating the use of modalityindependent elastography (MIE) [3-8] as a method for monitoring mechanical integrity to predict long term outcomes in NAC. Recently, we have utilized a Demons non-rigid image registration method that allows 3D elasticity reconstruction in abnormal tissue geometries, making it particularly amenable to the evaluation of breast cancer mechanical properties. While past work has reflected relative elasticity contrast ratios [3], this study improves upon that work by utilizing a known stiffness reference material within the reconstruction framework such that a stiffness map becomes an absolute measure. To test, a polyvinyl alcohol (PVA) cryogel phantom and a silicone rubber mock mouse tumor phantom were constructed with varying mechanical stiffness. Results showed that an absolute measure of stiffness could be obtained based on a reference value. This reference technique demonstrates the ability to generate accurate measurements of absolute stiffness to characterize response to NAC. These results support that `referenced MIE' has the potential to reliably differentiate absolute tumor stiffness with significant contrast from that of surrounding tissue. The use of referenced MIE to obtain absolute quantification of biomarkers is also translatable across length scales such that the characterization method is mechanics-consistent at the small animal and human application.

  2. Quantification and determination of spread mechanisms of bovine viral diarrhoea virus in blood and tissues from colostrum-deprived calves during an experimental acute infection induced by a non-cytopathic genotype 1 strain.

    PubMed

    Pedrera, M; Gómez-Villamandos, J C; Molina, V; Risalde, M A; Rodríguez-Sánchez, B; Sánchez-Cordón, P J

    2012-10-01

    To detect and monitor the sequential changes in virus levels, a reverse transcription quantitative real-time polymerase chain reaction assay using a TaqMan probe was carried out on frozen blood and tissues samples collected from calves experimentally infected with a non-cytopathic Bovine viral diarrhoea virus (BVDV) genotype 1 strain. Blood samples were collected among days 1-14 post-inoculation (p.i). On day 3 p.i, viral RNA was detected in blood samples from six of the eight inoculated animals. Viral RNA was detected in all remaining inoculated animals between 5 and 12 days p.i. The levels of viral RNA increased along the experiment, with a maximal peak between 6 and 9 days p.i. Analysis of virus load in tissues collected from calves euthanized on days 3, 6, 9 and 14 p.i displayed that BVDV was detected on day 3 p.i, being especially abundant in tonsils and ileocaecal valve, highlighting the role of tonsils as the main earliest viral replication sites as well as the principal source for virus spread to other lymphoid tissues and visceral organs. Coinciding with the highest viraemia levels, the highest viral loads were recorded at 9 days p.i. in tonsils, ileal lymph nodes, distal ileum and spleen, showing the main role of these secondary lymphoid organs in the pathogenic mechanisms of BVDV. However, virus levels in the liver and lung increased only towards the end of the infection. This fact could influence in the appearance of bovine respiratory diseases because of the capacity of BVDV for enhancing susceptibility to secondary infections.

  3. Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii.

    PubMed

    Jiang, Hucheng; Qian, Zhaojun; Lu, Wei; Ding, Huaiyu; Yu, Hongwei; Wang, Hui; Li, Jiale

    2015-09-08

    qRT-PCR is a widely used technique for rapid and accurate quantification of gene expression data. The use of reference genes for normalization of the expression levels is crucial for accuracy. Several studies have shown that there is no perfect reference gene that is appropriate for use in all experimental conditions, and research on suitable reference genes in red swamp crawfish (Procambarus clarkii) is particularly scarce. In this study, eight commonly used crustacean reference genes were chosen from P. clarkii transcriptome data and investigated as potential candidates for normalization of qRT-PCR data. Expression of these genes under different experimental conditions was examined by qRT-PCR, and the stability of their expression was evaluated using three commonly used statistical algorithms, geNorm, NormFinder and BestKeeper. A final comprehensive ranking determined that EIF and 18S were the optimal reference genes for expression data from different tissues, while TBP and EIF were optimal for expression data from different ovarian developmental stages. To our knowledge, this is the first systematic analysis of reference genes for normalization of qRT-PCR data in P. clarkii. These results will facilitate more accurate and reliable expression studies of this and other crustacean species.

  4. A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR

    PubMed Central

    Maier, Helena J.; Van Borm, Steven; Young, John R.; Fife, Mark

    2016-01-01

    Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. PMID:27537060

  5. Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

    PubMed

    Lin, Pengfei; Lan, Xiangli; Chen, Fenglei; Yang, Yanzhou; Jin, Yaping; Wang, Aihua

    2013-01-01

    The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS), NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

  6. Reference gene selection for quantitative real-time PCR normalization in Quercus suber.

    PubMed

    Marum, Liliana; Miguel, Andreia; Ricardo, Cândido P; Miguel, Célia

    2012-01-01

    The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

  7. Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer.

    PubMed

    El Hadi, Hicham; Abdellaoui-Maane, Imane; Kottwitz, Denise; El Amrani, Manal; Bouchoutrouch, Nadia; Qmichou, Zineb; Karkouri, Mehdi; ElAttar, Hicham; Errihani, Hassan; Fernandez, Pedro L; Bakri, Youssef; Sefrioui, Hassan; Moumen, Abdeladim

    2017-03-20

    Accurate measurement of Human epidermal growth factor receptor (HER2) gene expression is central for breast or stomach cancer therapy orientation and prognosis. The current standards testing methods for HER2 expression are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the current study, we explored the use of quantitative real time reverse transcription-PCR (RT-qPCR) as a potential method for the accurate relative quantification of the HER2 gene using formalin fixed paraffin embedded (FFPE) breast cancer biopsy samples. The main aim of the current study is to measure the level of concordance of RT-qPCR based quantification of HER2 overexpression with both IHC and FISH. Accordingly, an endogenous control gene (ECG) is required for this relative quantification and should ideally be expressed equivalently across tested samples. Stably expressed ECGs have been selected from a panel of seven genes using GenEx V6 software which is based on geNorm and NormFinder and statistical methods. Quantification of HER2 gene expression was performed by our RT-qPCR-based test and compared to the results obtained by both IHC and FISH methods. HER2 gene quantification using RT-qPCR test was normalized using the two ECGs (RPL30 and RPL37A) that were successfully identified and selected from a panel of seven genes as the most stable and reliable ECGs. We evaluated a total of 216 FFPE tissue samples from breast cancer patients. The results obtained with RT-qPCR in the current study were compared to both IHC and FISH data collected for the same patients. In addition to an internal evaluation, an external evaluation of this assay was also performed in a recognized pathology center in Europe (Clinic Barcelona Hospital Universitari, Spain) using 116 FFPE breast cancer tissue samples. The results demonstrated a high concordance between RT-qPCR and either IHC (98%) or FISH (72%) methods. Accordantly, the overall concordance was 85%. To our knowledge, this is the

  8. Pharmacokinetics of tildipirosin in porcine plasma, lung tissue, and bronchial fluid and effects of test conditions on in vitro activity against reference strains and field isolates of Actinobacillus pleuropneumoniae.

    PubMed

    Rose, M; Menge, M; Bohland, C; Zschiesche, E; Wilhelm, C; Kilp, S; Metz, W; Allan, M; Röpke, R; Nürnberger, M

    2013-04-01

    The pharmacokinetics of tildipirosin (Zuprevo(®) 40 mg/mL solution for injection for pigs), a novel 16-membered-ring macrolide for the treatment for swine respiratory disease (SRD), was investigated in studies collecting blood plasma and postmortem samples of lung tissue and bronchial fluid (BF) from swine. In view of factors influencing the in vitro activity of macrolides, and for the interpretation of tildipirosin pharmacokinetics in relation to minimum inhibitory concentrations (MIC), additional experiments were conducted to study the effects of pH, carbon dioxide-enriched atmosphere, buffers, and serum on tildipirosin MICs for various reference strains and Actinobacillus (A.) pleuropneumoniae field isolates. After single intramuscular (i.m.) injection at 4 mg/kg body weight, maximum plasma concentration (Cmax) was 0.9 μg/mL observed within 23 min (Tmax ). Mean residence time from the time of dosing to the time of last measurable concentration (MRTlast) and terminal half-life (T1/2) both were about 4 days. A dose-response relationship with no significant sex effect is observed for area under the plasma concentration-time curve from time 0 to the last sampling time with a quantifiable drug concentration (AUClast) over the range of doses up to 6 mg/kg. However, linear dose proportionality could not be proven with statistical methods. The time-concentration profile of tildipirosin in BF and lung far exceeded that in blood plasma. In lung, tildipirosin concentrations reached 3.1 μg/g at 2 h, peaked at 4.3 μg/g at day 1, and slowly declined to 0.8 μg/g at day 17. In BF, tildipirosin levels were 14.3, 7.0, and 6.5 μg/g at days 5, 10, and 14. T1/2 in lung was ∼7 days. Tildipirosin is rapidly and extensively distributed to the respiratory tract followed by slow elimination. Culture media pH and carbon dioxide-enriched atmosphere (CO2 -EA) had a marked impact on in vitro activity of tildipirosin in reference strains of various rapidly growing aerobic and

  9. Advancing agricultural greenhouse gas quantification*

    NASA Astrophysics Data System (ADS)

    Olander, Lydia; Wollenberg, Eva; Tubiello, Francesco; Herold, Martin

    2013-03-01

    1. Introduction Better information on greenhouse gas (GHG) emissions and mitigation potential in the agricultural sector is necessary to manage these emissions and identify responses that are consistent with the food security and economic development priorities of countries. Critical activity data (what crops or livestock are managed in what way) are poor or lacking for many agricultural systems, especially in developing countries. In addition, the currently available methods for quantifying emissions and mitigation are often too expensive or complex or not sufficiently user friendly for widespread use. The purpose of this focus issue is to capture the state of the art in quantifying greenhouse gases from agricultural systems, with the goal of better understanding our current capabilities and near-term potential for improvement, with particular attention to quantification issues relevant to smallholders in developing countries. This work is timely in light of international discussions and negotiations around how agriculture should be included in efforts to reduce and adapt to climate change impacts, and considering that significant climate financing to developing countries in post-2012 agreements may be linked to their increased ability to identify and report GHG emissions (Murphy et al 2010, CCAFS 2011, FAO 2011). 2. Agriculture and climate change mitigation The main agricultural GHGs—methane and nitrous oxide—account for 10%-12% of anthropogenic emissions globally (Smith et al 2008), or around 50% and 60% of total anthropogenic methane and nitrous oxide emissions, respectively, in 2005. Net carbon dioxide fluxes between agricultural land and the atmosphere linked to food production are relatively small, although significant carbon emissions are associated with degradation of organic soils for plantations in tropical regions (Smith et al 2007, FAO 2012). Population growth and shifts in dietary patterns toward more meat and dairy consumption will lead to

  10. A telepathology based Virtual Reference and Certification Centre for DNA image cytometry.

    PubMed

    Haroske, G; Giroud, F; Kunze, K D; Meyer, W

    2000-01-01

    An increasing need for flexible consultation between pathologists, including the application of fast evolving supplementary technologies, has been identified during the last years. Although pathology is already one of the most advanced application of telemedicine there is more to come from the fast evolution towards computerized microscope image analysis: A reproducible quantification of measurable descriptors of the lesions in cells and tissues (so-called biological markers) is an indispensable adjunct to routine diagnostic application. Among such quantitative methods DNA image cytometry is increasingly applied by pathologists for assistance in diagnostics. As for other pathological issues, too, a reference center for the clinical application of DNA image cytometry might be therefore of utmost value for pathologists using that method. Based on advanced telematic technologies, a Virtual Reference and Certification Center (VRCC) could be installed for certifying the cytometry hardware and software, the analytical procedures, and the basic interpretation of the results. It will be designed to be operated as a non-attended service, based on quantification servers accessible via Internet round the clock. The VRCC will supply appropriate standardization and normalization materials and run a GroupWare platform for consensus making by experts.

  11. Identification of reliable reference genes for qRT-PCR studies of the developing mouse mammary gland

    PubMed Central

    van de Moosdijk, Anoeska Agatha Alida; van Amerongen, Renée

    2016-01-01

    Cell growth and differentiation are often driven by subtle changes in gene expression. Many challenges still exist in detecting these changes, particularly in the context of a complex, developing tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) allows relatively high-throughput evaluation of multiple genes and developmental time points. Proper quantification of gene expression levels by qRT-PCR requires normalization to one or more reference genes. Traditionally, these genes have been selected based on their presumed “housekeeping” function, with the implicit assumption that they are stably expressed over the entire experimental set. However, this is rarely tested empirically. Here we describe the identification of novel reference genes for the mouse mammary gland based on their stable expression in published microarray datasets. We compared eight novel candidate reference genes (Arpc3, Clock, Ctbp1, Phf7, Prdx1, Sugp2, Taf11 and Usp7) to eight traditional ones (18S, Actb, Gapdh, Hmbs, Hprt, Rpl13a, Sdha and Tbp) and analysed all genes for stable expression in the mouse mammary gland from pre-puberty to adulthood using four different algorithms (GeNorm, DeltaCt, BestKeeper and NormFinder). Prdx1, Phf7 and Ctbp1 were validated as novel and reliable, tissue-specific reference genes that outperform traditional reference genes in qRT-PCR studies of postnatal mammary gland development. PMID:27752147

  12. Wrappers, Aspects, Quantification and Events

    NASA Technical Reports Server (NTRS)

    Filman, Robert E.

    2005-01-01

    Talk overview: Object infrastructure framework (OIF). A system development to simplify building distributed applications by allowing independent implementation of multiple concern. Essence and state of AOP. Trinity. Quantification over events. Current work on a generalized AOP technology.

  13. Quantification of confocal images of biofilms grown on irregular surfaces

    PubMed Central

    Ross, Stacy Sommerfeld; Tu, Mai Han; Falsetta, Megan L.; Ketterer, Margaret R.; Kiedrowski, Megan R.; Horswill, Alexander R.; Apicella, Michael A.; Reinhardt, Joseph M.; Fiegel, Jennifer

    2014-01-01

    Bacterial biofilms grow on many types of surfaces, including flat surfaces such as glass and metal and irregular surfaces such as rocks, biological tissues and polymers. While laser scanning confocal microscopy can provide high-resolution images of biofilms grown on any surface, quantification of biofilm-associated bacteria is currently limited to bacteria grown on flat surfaces. This can limit researchers studying irregular surfaces to qualitative analysis or quantification of only the total bacteria in an image. In this work, we introduce a new algorithm called modified connected volume filtration (MCVF) to quantify bacteria grown on top of an irregular surface that is fluorescently labeled or reflective. Using the MCVF algorithm, two new quantification parameters are introduced. The modified substratum coverage parameter enables quantification of the connected-biofilm bacteria on top of the surface and on the imaging substratum. The utility of MCVF and the modified substratum coverage parameter were shown with Pseudomonas aeruginosa and Staphylococcus aureus biofilms grown on human airway epithelial cells. A second parameter, the percent association, provides quantified data on the colocalization of the bacteria with a labeled component, including bacteria within a labeled tissue. The utility of quantifying the bacteria associated with the cell cytoplasm was demonstrated with Neisseria gonorrhoeae biofilms grown on cervical epithelial cells. This algorithm provides more flexibility and quantitative ability to researchers studying biofilms grown on a variety of irregular substrata. PMID:24632515

  14. Quantification of human responses

    NASA Technical Reports Server (NTRS)

    Steinlage, R. C.; Gantner, T. E.; Lim, P. Y. W.

    1992-01-01

    Human perception is a complex phenomenon which is difficult to quantify with instruments. For this reason, large panels of people are often used to elicit and aggregate subjective judgments. Print quality, taste, smell, sound quality of a stereo system, softness, and grading Olympic divers and skaters are some examples of situations where subjective measurements or judgments are paramount. We usually express what is in our mind through language as a medium but languages are limited in available choices of vocabularies, and as a result, our verbalizations are only approximate expressions of what we really have in mind. For lack of better methods to quantify subjective judgments, it is customary to set up a numerical scale such as 1, 2, 3, 4, 5 or 1, 2, 3, ..., 9, 10 for characterizing human responses and subjective judgments with no valid justification except that these scales are easy to understand and convenient to use. But these numerical scales are arbitrary simplifications of the complex human mind; the human mind is not restricted to such simple numerical variations. In fact, human responses and subjective judgments are psychophysical phenomena that are fuzzy entities and therefore difficult to handle by conventional mathematics and probability theory. The fuzzy mathematical approach provides a more realistic insight into understanding and quantifying human responses. This paper presents a method for quantifying human responses and subjective judgments without assuming a pattern of linear or numerical variation for human responses. In particular, quantification and evaluation of linguistic judgments was investigated.

  15. Uncertainty Quantification in Aeroelasticity

    NASA Astrophysics Data System (ADS)

    Beran, Philip; Stanford, Bret; Schrock, Christopher

    2017-01-01

    Physical interactions between a fluid and structure, potentially manifested as self-sustained or divergent oscillations, can be sensitive to many parameters whose values are uncertain. Of interest here are aircraft aeroelastic interactions, which must be accounted for in aircraft certification and design. Deterministic prediction of these aeroelastic behaviors can be difficult owing to physical and computational complexity. New challenges are introduced when physical parameters and elements of the modeling process are uncertain. By viewing aeroelasticity through a nondeterministic prism, where key quantities are assumed stochastic, one may gain insights into how to reduce system uncertainty, increase system robustness, and maintain aeroelastic safety. This article reviews uncertainty quantification in aeroelasticity using traditional analytical techniques not reliant on computational fluid dynamics; compares and contrasts this work with emerging methods based on computational fluid dynamics, which target richer physics; and reviews the state of the art in aeroelastic optimization under uncertainty. Barriers to continued progress, for example, the so-called curse of dimensionality, are discussed.

  16. Quantification of meat proportions by measuring DNA contents in raw and boiled sausages using matrix-adapted calibrators and multiplex real-time PCR.

    PubMed

    Köppel, René; Eugster, Albert; Ruf, Jürg; Rentsch, Jürg

    2012-01-01

    The quantification of meat proportions in raw and boiled sausage according to the recipe was evaluated using three different calibrators. To measure the DNA contents from beef, pork, sheep (mutton), and horse, a tetraplex real-time PCR method was applied. Nineteen laboratories analyzed four meat products each made of different proportions of beef, pork, sheep, and horse meat. Three kinds of calibrators were used: raw and boiled sausages of known proportions ranging from 1 to 55% of meat, and a dilution series of DNA from muscle tissue. In general, results generated using calibration sausages were more accurate than those resulting from the use of DNA from muscle tissue, and exhibited smaller measurement uncertainties. Although differences between uses of raw and boiled calibration sausages were small, the most precise and accurate results were obtained by calibration with fine-textured boiled reference sausages.

  17. Validation of endogenous control reference genes for normalizing gene expression studies in endometrial carcinoma.

    PubMed

    Ayakannu, Thangesweran; Taylor, Anthony H; Willets, Jonathon M; Brown, Laurence; Lambert, David G; McDonald, John; Davies, Quentin; Moss, Esther L; Konje, Justin C

    2015-09-01

    Real-time quantitative RT-PCR (qRT-PCR) is a powerful technique used for the relative quantification of target genes, using reference (housekeeping) genes for normalization to ensure the generation of accurate and robust data. A systematic examination of the suitability of endogenous reference genes for gene expression studies in endometrial cancer tissues is absent. The aims of this study were therefore to identify and evaluate from the thirty-two possible reference genes from a TaqMan(®) array panel their suitability as an internal control gene. The mathematical software packages geNorm qBasePLUS identified Pumilio homolog 1 (Drosophila) (PUM1), ubiquitin C (UBC), phosphoglycerate kinase (PGK1), mitochondrial ribosomal protein L19 (MRPL19) and peptidylpropyl isomerase A (cyclophilin A) (PPIA) as the best reference gene combination, whilst NormFinder identified MRPL19 as the best single reference gene, with importin 8 (IPO8) and PPIA being the best combination of two reference genes. BestKeeper ranked MRPL19 as the most stably expressed gene. In addition, the study was validated by examining the relative expression of a test gene, which encodes the cannabinoid receptor 1 (CB1). A significant difference in CB1 mRNA expression between malignant and normal endometrium using MRPL19, PPIA, and IP08 in combination was observed. The use of MRPL19, IPO8 and PPIA was identified as the best reference gene combination for the normalization of gene expression levels in endometrial carcinoma. This study demonstrates that the arbitrary selection of endogenous control reference genes for normalization in qRT-PCR studies of endometrial carcinoma, without validation, risks the production of inaccurate data and should therefore be discouraged.

  18. Ultra-trace analysis of platinum in human tissue samples.

    PubMed

    Rudolph, Elisabeth; Hann, Stephan; Stingeder, Gerhard; Reiter, Christian

    2005-08-01

    Background levels of platinum were determined in human autopsy tissues taken from five individuals. The investigated specimens were lung, liver and kidney. Sample preparation involved microwave digestion followed by an open vessel treatment. Inductively-coupled plasma sector field mass spectrometry (ICP-SFMS) was applied in combination with an ultrasonic nebulization/membrane desolvation system for sample introduction. Isotope dilution analysis was employed for accurate quantification of platinum. Excellent procedural detection limits (3 s validation) of 20, 20 and 34 pg g(-1) dry weight were obtained for lung, liver and kidney tissue, respectively. Due to the lack of appropriate biological reference material, road dust (BCR-723) was used for method validation. Platinum levels ranging between 0.03 and 1.42 ng g(-1) were determined in the investigated samples. The platinum concentrations observed in human lung tissue may reflect the increasing atmospheric background levels of platinum originating from car catalysts. The presence of platinum in kidney and liver tissue samples clearly indicates the bioavailability of the element.

  19. Quantification of jasmonic and salicylic acids in rice seedling leaves.

    PubMed

    Cho, Kyoungwon; Han, Oksoo; Tamogami, Shigeru; Shibato, Junko; Kubo, Akihiro; Agrawal, Ganesh Kumar; Rakwal, Randeep

    2013-01-01

    Jasmonic acid (JA) and salicylic acid (SA) are critical signaling components involved in various aspects of plant growth, development, and defense. Their constitutive levels vary from plant to plant and also from tissue to tissue within the same plant. Moreover, their quantitative levels change when plant is exposed to biotic and abiotic stresses. To better understand the JA- and SA-mediated signaling and metabolic pathways, it is important to precisely quantify their levels in plants/tissues/organs. However, their extraction and quantification are not trivial and still technically challenging. An effort has been made in various laboratories to develop a simple and standard procedure that can be utilized for quantification of JA and SA. Here, we present the experimental procedure and our decade of experience on extracting and quantifying them in an absolute manner in leaves of rice seedlings. We must mention that this method has been applied to both monocotyledonous and dicotyledonous plants for absolute quantification of JA and SA. As collaboration is the key towards rapid progress in science and technology, we are always open to sharing our experience in this field with any active research group with an aim to improve the procedure further and eventually to connect the importance of their (JA and SA) quantitative levels with networks of signaling and metabolic pathways in plants.

  20. Reference Frames and Relativity.

    ERIC Educational Resources Information Center

    Swartz, Clifford

    1989-01-01

    Stresses the importance of a reference frame in mechanics. Shows the Galilean transformation in terms of relativity theory. Discusses accelerated reference frames and noninertial reference frames. Provides examples of reference frames with diagrams. (YP)

  1. Reference materials for new psychoactive substances.

    PubMed

    Archer, Roland P; Treble, Ric; Williams, Keith

    2011-01-01

    Historically, the appearance of new psychoactive materials (and hence the requirement for new reference standards) has been relatively slow. This position has now changed, with 101 new psychoactive substances reported to EMCDDA-Europol since 2006. The newly reported materials, and associated metabolites, require properly certified reference materials to permit reliable identification and quantification. The traditional approach and timescales of reference material production and certification are being increasingly challenged by the appearance of these new substances. Reference material suppliers have to adopt new strategies to meet the needs of laboratories. This situation is particularly challenging for toxicology standards as the metabolism of many of these substances is initially unknown. Reference material production often involves synthesis from first principles. While it is possible to synthesis these materials, there can be significant difficulties, from synthetic complexities through to the need to use controlled materials. These issues are examined through a discussion of the synthesis of cathinones. Use of alternative sources, including pharmaceutical impurity materials or internet sourced products, as starting materials for conversion into appropriately certified reference materials are also discussed. The sudden appearance and sometimes brief lifetime in the market place of many of these novel legal highs or research chemicals present commercial difficulties for reference material producers. The need for collaboration at all levels is highlighted as essential to rapid identification of requirements for new reference materials. National or international commissioning or support may also be required to permit reference material producers to recover their development costs.

  2. A rapid digestion method for analysis of nickel compounds in tissue by electrothermal atomic absorption spectrometry.

    PubMed

    Benson, J M; Eidson, A F; Hanson, R L; Henderson, R F; Hobbs, C H

    1989-08-01

    Quantification of nickel in animal soft tissue is of toxicological interest. A digestion method applying the use of microwave ovens for irradiating samples in Teflon digesters was developed. An acid mixture containing nitric acid (16 M, 1.0 ml g-1 tissue), hydrochloric acid (6 M, 0.5 ml g-1 tissue) and H2O2 (30%, 1.0 ml g-1 tissue) and irradiation at 600 W for 5 min were required for complete dissolution of tissue matrices and nickel compounds. Analyses of Ni in National Bureau of Standards Reference Material 1566 oyster tissue gave 0.87 +/- 0.24 micrograms g 1(mean +/- SD, n = 5), which was in agreement with the NBS certified value of 1.03 +/- 0.19 micrograms g-1. Recoveries of 1-300 micrograms Ni added as nickel sulfate (highly soluble), nickel subsulfide (moderately soluble in biological fluids and acid) or nickel oxide (green high-temperature oxide, low solubility in biological fluids and acid) to lung, liver, lymph node and kidney were quantitative, except in the case of nickel sulfate added to kidney, where recovery was less than quantitative for 1-10 micrograms Ni. The method appears effective for digestion of a variety of tissues requiring Ni analyses.

  3. Noninvasive bi-graphical analysis for the quantification of slowly reversible radioligand binding

    NASA Astrophysics Data System (ADS)

    Seo, Seongho; Kim, Su Jin; Yoo, Hye Bin; Lee, Jee-Young; Kyeong Kim, Yu; Lee, Dong Soo; Zhou, Yun; Lee, Jae Sung

    2016-09-01

    In this paper, we presented a novel reference-region-based (noninvasive) bi-graphical analysis for the quantification of a reversible radiotracer binding that may be too slow to reach relative equilibrium (RE) state during positron emission tomography (PET) scans. The proposed method indirectly implements the noninvasive Logan plot, through arithmetic combination of the parameters of two other noninvasive methods and the apparent tissue-to-plasma efflux rate constant for the reference region (k2\\prime ). We investigated its validity and statistical properties, by performing a simulation study with various noise levels and k2\\prime values, and also evaluated its feasibility for [18F]FP-CIT PET in human brain. The results revealed that the proposed approach provides distribution volume ratio estimation comparable to the Logan plot at low noise levels while improving underestimation caused by non-RE state differently depending on k2\\prime . Furthermore, the proposed method was able to avoid noise-induced bias of the Logan plot, and the variability of its results was less dependent on k2\\prime than the Logan plot. Therefore, this approach, without issues related to arterial blood sampling given a pre-estimate of k2\\prime (e.g. population-based), could be useful in parametric image generation for slow kinetic tracers staying in a non-RE state within a PET scan.

  4. Micro-RNA quantification using DNA polymerase and pyrophosphate quantification.

    PubMed

    Yu, Hsiang-Ping; Hsiao, Yi-Ling; Pan, Hung-Yin; Huang, Chih-Hung; Hou, Shao-Yi

    2011-12-15

    A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20-100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays.

  5. Dual-wavelength excitation for fluorescence-based quantification of zinc protoporphyrin IX and protoporphyrin IX in whole blood.

    PubMed

    Hennig, Georg; Gruber, Christian; Vogeser, Michael; Stepp, Herbert; Dittmar, Stephan; Sroka, Ronald; Brittenham, Gary M

    2014-07-01

    Quantification of erythrocyte zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PPIX), individually or jointly, is useful for the diagnostic evaluation of iron deficiency, iron-restricted erythropoiesis, lead exposure, and porphyrias. A method for simultaneous quantification of ZnPP and PPIX in unwashed blood samples is described, using dual-wavelength excitation to effectively eliminate background fluorescence from other blood constituents. In blood samples from 35 subjects, the results of the dual-wavelength excitation method and a reference high performance liquid chromatography (HPLC) assay were closely correlated both for ZnPP (rs = 0.943, p < 0.0001; range 37-689 μmol ZnPP/mol heme, 84-1238 nmol/L) and for PPIX (rs = 0.959, p < 0.0001; range 42-4212 μmol PPIX/mol heme, 93-5394 nmol/L). In addition, for ZnPP, the proposed method is compared with conventional single-wavelength excitation and with commercial front-face fluorimetry of washed erythrocytes and whole blood. We hypothesize that dual-wavelength excitation fluorimetry will provide a new approach to the suppression of background fluorescence in blood and tissue measurements of ZnPP and PPIX.

  6. MAMA Software Features: Quantification Verification Documentation-1

    SciTech Connect

    Ruggiero, Christy E.; Porter, Reid B.

    2014-05-21

    This document reviews the verification of the basic shape quantification attributes in the MAMA software against hand calculations in order to show that the calculations are implemented mathematically correctly and give the expected quantification results.

  7. Droplet digital PCR for absolute quantification of pathogens.

    PubMed

    Gutiérrez-Aguirre, Ion; Rački, Nejc; Dreo, Tanja; Ravnikar, Maja

    2015-01-01

    The recent advent of different digital PCR (dPCR) platforms is enabling the expansion of this technology for research and diagnostic applications worldwide. The main principle of dPCR, as in other PCR-based methods including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions which is followed by a real time or end point detection of the amplification. The distribution of target sequences into partitions is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. dPCR has also shown higher resilience to inhibitors in a number of different types of samples. In this chapter we describe the droplet digital PCR (ddPCR) workflow for the detection and quantification of pathogens using the droplet digital Bio-Rad platform QX100. We present as an example the quantification of the quarantine plant pathogenic bacterium, Erwinia amylovora.

  8. Partial volume effect modeling for segmentation and tissue classification of brain magnetic resonance images: A review.

    PubMed

    Tohka, Jussi

    2014-11-28

    Quantitative analysis of magnetic resonance (MR) brain images are facilitated by the development of automated segmentation algorithms. A single image voxel may contain of several types of tissues due to the finite spatial resolution of the imaging device. This phenomenon, termed partial volume effect (PVE), complicates the segmentation process, and, due to the complexity of human brain anatomy, the PVE is an important factor for accurate brain structure quantification. Partial volume estimation refers to a generalized segmentation task where the amount of each tissue type within each voxel is solved. This review aims to provide a systematic, tutorial-like overview and categorization of methods for partial volume estimation in brain MRI. The review concentrates on the statistically based approaches for partial volume estimation and also explains differences to other, similar image segmentation approaches.

  9. Reference gene selection for quantitative real-time PCR in Solanum lycopersicum L. inoculated with the mycorrhizal fungus Rhizophagus irregularis.

    PubMed

    Fuentes, Alejandra; Ortiz, Javier; Saavedra, Nicolás; Salazar, Luis A; Meneses, Claudio; Arriagada, Cesar

    2016-04-01

    The gene expression stability of candidate reference genes in the roots and leaves of Solanum lycopersicum inoculated with arbuscular mycorrhizal fungi was investigated. Eight candidate reference genes including elongation factor 1 α (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), protein phosphatase 2A (PP2Acs), ribosomal protein L2 (RPL2), β-tubulin (TUB), ubiquitin (UBI) and actin (ACT) were selected, and their expression stability was assessed to determine the most stable internal reference for quantitative PCR normalization in S. lycopersicum inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. The stability of each gene was analysed in leaves and roots together and separated using the geNorm and NormFinder algorithms. Differences were detected between leaves and roots, varying among the best-ranked genes depending on the algorithm used and the tissue analysed. PGK, TUB and EF1 genes showed higher stability in roots, while EF1 and UBI had higher stability in leaves. Statistical algorithms indicated that the GAPDH gene was the least stable under the experimental conditions assayed. Then, we analysed the expression levels of the LePT4 gene, a phosphate transporter whose expression is induced by fungal colonization in host plant roots. No differences were observed when the most stable genes were used as reference genes. However, when GAPDH was used as the reference gene, we observed an overestimation of LePT4 expression. In summary, our results revealed that candidate reference genes present variable stability in S. lycopersicum arbuscular mycorrhizal symbiosis depending on the algorithm and tissue analysed. Thus, reference gene selection is an important issue for obtaining reliable results in gene expression quantification.

  10. Biological and environmental reference materials: Update 1996

    NASA Astrophysics Data System (ADS)

    Roelandts, Iwan

    1997-07-01

    The present column lists additional biological and environmental reference samples. Organs, tissues, body fluids, plant materials, foods, fuels, ashes, dusts, particulate matter, gas mixtures, oils, soils, sediments, sludges and waters have been considered. Three tables are included that provide an easy-to-use survey. The following information is covered: the name of the material, the sample code, the producer, the reference to certification, the names and addresses of the suppliers from whom the reference material may be obtained, and specific remarks.

  11. Reference Service Policy Statement.

    ERIC Educational Resources Information Center

    Young, William F.

    This reference service policy manual provides general guidelines to encourage reference service of the highest possible quality and to insure uniform practice. The policy refers only to reference service in the University Libraries and is intended for use in conjunction with other policies and procedures issued by the Reference Services Division.…

  12. Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

    PubMed

    Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

    2016-02-01

    Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis.

  13. Automatic quantification of neo-vasculature from micro-CT

    NASA Astrophysics Data System (ADS)

    Mallya, Yogish; Narayanan, A. K.; Zagorchev, Lyubomir

    2009-02-01

    Angiogenesis is the process of formation of new blood vessels as outgrowths of pre-existing ones. It occurs naturally during development, tissue repair, and abnormally in pathologic diseases such as cancer. It is associated with proliferation of blood vessels/tubular sprouts that penetrate deep into tissues to supply nutrients and remove waste products. The process starts with migration of endothelial cells. As the cells move towards the target area they form small tubular sprouts recruited from the parent vessel. The sprouts grow in length due to migration, proliferation, and recruitment of new endothelial cells and the process continues until the target area becomes fully vascular. Accurate quantification of sprout formation is very important for evaluation of treatments for ischemia as well as angiogenesis inhibitors and plays a key role in the battle against cancer. This paper presents a technique for automatic quantification of newly formed blood vessels from Micro-CT volumes of tumor samples. A semiautomatic technique based on interpolation of Bezier curves was used to segment out the cancerous growths. Small vessels as determined by their diameter within the segmented tumors were enhanced and quantified with a multi-scale 3-D line detection filter. The same technique can be easily extended for quantification of tubular structures in other 3-D medical imaging modalities. Experimental results are presented and discussed.

  14. Evaluation of reference genes for real-time PCR studies of Brazilian Somalis sheep infected by gastrointestinal nematodes

    PubMed Central

    2010-01-01

    Precise normalization with reference genes is necessary, in order to obtain reliable relative expression data in response to gastrointestinal nematode infection. By using sheep from temperate regions as models, three reference genes, viz., ribosomal protein LO (RPLO), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and succinate dehydrogenase complex subunit A (SDHA), were investigated in the abomasum, abomasal lymph nodes and small intestine of Brazilian Somalis sheep, either resistant or susceptible to gastrointestinal nematodes infections. Real time PCR was carried out by using SYBR Green I dye, and gene stability was tested by geNorm. RPLO was an ideal reference gene, since its expression was constant across treatments, presented lower variation, and was ranked as the most stable in abomasum and lymph node tissues. On the other hand, SDHA was the most stable in the small intestine followed by RPLO and GAPDH. These findings demonstrate the importance of correctly choosing reference genes prior to relative quantification. In addition, we determined that reference genes used in sheep from temperate regions, when properly tested, can be applied in animals from tropical regions such as the Brazilian Somalis sheep. PMID:21637421

  15. Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions.

    PubMed

    Nakayama, T J; Rodrigues, F A; Neumaier, N; Marcelino-Guimarães, F C; Farias, J R B; de Oliveira, M C N; Borém, A; de Oliveira, A C B; Emygdio, B M; Nepomuceno, A L

    2014-02-13

    Quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful tool used to measure gene expression. However, because of its high sensitivity, the method is strongly influenced by the quality and concentration of the template cDNA and by the amplification efficiency. Relative quantification is an effective strategy for correcting random and systematic errors by using the expression level of reference gene(s) to normalize the expression level of the genes of interest. To identify soybean reference genes for use in studies of flooding stress, we compared 5 candidate reference genes (CRGs) with the NormFinder and GeNorm programs to select the best internal control. The expression stability of the CRGs was evaluated in root tissues from soybean plants subjected to hypoxic conditions. Elongation factor 1-beta and actin-11 were identified as the most appropriate genes for RT-qPCR normalization by both the NormFinder and GeNorm analyses. The expression profiles of the genes for alcohol dehydrogenase 1, sucrose synthase 4, and ascorbate peroxidase 2 were analyzed by comparing different normalizing combinations (including no normalization) of the selected reference genes. Here, we have identified potential genes for use as references for RT-qPCR normalization in experiments with soybean roots growing in O2-depleted environments, such as flooding-stressed plants.

  16. Selection of suitable reference genes for assessing gene expression in pearl millet under different abiotic stresses and their combinations.

    PubMed

    Shivhare, Radha; Lata, Charu

    2016-03-14

    Pearl millet [Pennisetum glaucum (L.) R. Br.] a widely used grain and forage crop, is grown in areas frequented with one or more abiotic stresses, has superior drought and heat tolerance and considered a model crop for stress tolerance studies. Selection of suitable reference genes for quantification of target stress-responsive gene expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of improved stress tolerance. For precise normalization of gene expression data in pearl millet, ten candidate reference genes were examined in various developmental tissues as well as under different individual abiotic stresses and their combinations at 1 h (early) and 24 h (late) of stress using geNorm, NormFinder and RefFinder algorithms. Our results revealed EF-1α and UBC-E2 as the best reference genes across all samples, the specificity of which was confirmed by assessing the relative expression of a PgAP2 like-ERF gene that suggested use of these two reference genes is sufficient for accurate transcript normalization under different stress conditions. To our knowledge this is the first report on validation of reference genes under different individual and multiple abiotic stresses in pearl millet. The study can further facilitate fastidious discovery of stress-tolerance genes in this important stress-tolerant crop.

  17. Selection of suitable reference genes for assessing gene expression in pearl millet under different abiotic stresses and their combinations

    PubMed Central

    Shivhare, Radha; Lata, Charu

    2016-01-01

    Pearl millet [Pennisetum glaucum (L.) R. Br.] a widely used grain and forage crop, is grown in areas frequented with one or more abiotic stresses, has superior drought and heat tolerance and considered a model crop for stress tolerance studies. Selection of suitable reference genes for quantification of target stress-responsive gene expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of improved stress tolerance. For precise normalization of gene expression data in pearl millet, ten candidate reference genes were examined in various developmental tissues as well as under different individual abiotic stresses and their combinations at 1 h (early) and 24 h (late) of stress using geNorm, NormFinder and RefFinder algorithms. Our results revealed EF-1α and UBC-E2 as the best reference genes across all samples, the specificity of which was confirmed by assessing the relative expression of a PgAP2 like-ERF gene that suggested use of these two reference genes is sufficient for accurate transcript normalization under different stress conditions. To our knowledge this is the first report on validation of reference genes under different individual and multiple abiotic stresses in pearl millet. The study can further facilitate fastidious discovery of stress-tolerance genes in this important stress-tolerant crop. PMID:26972345

  18. Towards future reference systems for GM analysis.

    PubMed

    Trapmann, Stefanie; Corbisier, Philippe; Schimmel, Heinz; Emons, Hendrik

    2010-03-01

    Despite the fact that the measurement unit for the quantification of GMOs in food and feed products has not yet been unambiguously agreed upon in Europe, international trade requires reliable GMO analysis measuring comparably the GMO content of products. The two reference systems, based either on mass fractions or on copy number ratios, and their metrological traceability chains are presented and discussed. It is concluded that, properly established and expressed, measurement results in copy number ratios can provide a metrologically sound reference system. In this case, certified reference materials used for calibration and quality control can be independent of each other and the uncertainty derived from calibration can correctly be included in the overall uncertainty of the GMO measurement. However, further efforts are required to establish this metrological system.

  19. Absolute quantification of myocardial blood flow.

    PubMed

    Yoshinaga, Keiichiro; Manabe, Osamu; Tamaki, Nagara

    2016-07-21

    With the increasing availability of positron emission tomography (PET) myocardial perfusion imaging, the absolute quantification of myocardial blood flow (MBF) has become popular in clinical settings. Quantitative MBF provides an important additional diagnostic or prognostic information over conventional visual assessment. The success of MBF quantification using PET/computed tomography (CT) has increased the demand for this quantitative diagnostic approach to be more accessible. In this regard, MBF quantification approaches have been developed using several other diagnostic imaging modalities including single-photon emission computed tomography, CT, and cardiac magnetic resonance. This review will address the clinical aspects of PET MBF quantification and the new approaches to MBF quantification.

  20. Survey and Evaluate Uncertainty Quantification Methodologies

    SciTech Connect

    Lin, Guang; Engel, David W.; Eslinger, Paul W.

    2012-02-01

    The Carbon Capture Simulation Initiative (CCSI) is a partnership among national laboratories, industry and academic institutions that will develop and deploy state-of-the-art computational modeling and simulation tools to accelerate the commercialization of carbon capture technologies from discovery to development, demonstration, and ultimately the widespread deployment to hundreds of power plants. The CCSI Toolset will provide end users in industry with a comprehensive, integrated suite of scientifically validated models with uncertainty quantification, optimization, risk analysis and decision making capabilities. The CCSI Toolset will incorporate commercial and open-source software currently in use by industry and will also develop new software tools as necessary to fill technology gaps identified during execution of the project. The CCSI Toolset will (1) enable promising concepts to be more quickly identified through rapid computational screening of devices and processes; (2) reduce the time to design and troubleshoot new devices and processes; (3) quantify the technical risk in taking technology from laboratory-scale to commercial-scale; and (4) stabilize deployment costs more quickly by replacing some of the physical operational tests with virtual power plant simulations. The goal of CCSI is to deliver a toolset that can simulate the scale-up of a broad set of new carbon capture technologies from laboratory scale to full commercial scale. To provide a framework around which the toolset can be developed and demonstrated, we will focus on three Industrial Challenge Problems (ICPs) related to carbon capture technologies relevant to U.S. pulverized coal (PC) power plants. Post combustion capture by solid sorbents is the technology focus of the initial ICP (referred to as ICP A). The goal of the uncertainty quantification (UQ) task (Task 6) is to provide a set of capabilities to the user community for the quantification of uncertainties associated with the carbon

  1. Flow Cytometry Analyses of Adipose Tissue Macrophages

    PubMed Central

    Cho, Kae Won; Morris, David L.; Lumeng, Carey N.

    2014-01-01

    Within adipose tissue, multiple leukocyte interactions contribute to metabolic homeostasis in health as well as to the pathogenesis of insulin resistance with obesity. Adipose tissue macrophages (ATMs) are the predominant leukocyte population in fat and contribute to obesity-induced inflammation. Characterization of ATMs and other leukocytes in the stromal vascular fraction from fat has benefited from the use of flow cytometry and flow-assisted cell sorting techniques. These methods permit the immunophenotyping, quantification, and purification of these unique cell populations from multiple adipose tissue depots in rodents and humans. Proper isolation, quantification, and characterization of ATM phenotypes are critical for understanding their role in adipose tissue function and obesity-induced metabolic diseases. Here, we present the flow cytometry protocols for phenotyping ATMs in lean and obese mice employed by our laboratory. PMID:24480353

  2. Quantification of breast density using dual-energy mammography with liquid phantom calibration.

    PubMed

    Lam, Alfonso R; Ding, Huanjun; Molloi, Sabee

    2014-07-21

    Breast density is a widely recognized potential risk factor for breast cancer. However, accurate quantification of breast density is a challenging task in mammography. The current use of plastic breast-equivalent phantoms for calibration provides limited accuracy in dual-energy mammography due to the chemical composition of the phantom. We implemented a breast-equivalent liquid phantom for dual-energy calibration in order to improve the accuracy of breast density measurement. To design these phantoms, three liquid compounds were chosen: water, isopropyl alcohol, and glycerol. Chemical compositions of glandular and adipose tissues, obtained from NIST database, were used as reference materials. Dual-energy signal of the liquid phantom at different breast densities (0% to 100%) and thicknesses (1 to 8 cm) were simulated. Glandular and adipose tissue thicknesses were estimated from a higher order polynomial of the signals. Our results indicated that the linear attenuation coefficients of the breast-equivalent liquid phantoms match those of the target material. Comparison between measured and known breast density data shows a linear correlation with a slope close to 1 and a non-zero intercept of 7%, while plastic phantoms showed a slope of 0.6 and a non-zero intercept of 8%. Breast density results derived from the liquid calibration phantoms showed higher accuracy than those derived from the plastic phantoms for different breast thicknesses and various tube voltages. We performed experimental phantom studies using liquid phantoms and then compared the computed breast density with those obtained using a bovine tissue model. The experimental data and the known values were in good correlation with a slope close to 1 (∼1.1). In conclusion, our results indicate that liquid phantoms are a reliable alternative for calibration in dual-energy mammography and better reproduce the chemical properties of the target material.

  3. Quantification of breast density using dual-energy mammography with liquid phantom calibration

    NASA Astrophysics Data System (ADS)

    Lam, Alfonso R.; Ding, Huanjun; Molloi, Sabee

    2014-07-01

    Breast density is a widely recognized potential risk factor for breast cancer. However, accurate quantification of breast density is a challenging task in mammography. The current use of plastic breast-equivalent phantoms for calibration provides limited accuracy in dual-energy mammography due to the chemical composition of the phantom. We implemented a breast-equivalent liquid phantom for dual-energy calibration in order to improve the accuracy of breast density measurement. To design these phantoms, three liquid compounds were chosen: water, isopropyl alcohol, and glycerol. Chemical compositions of glandular and adipose tissues, obtained from NIST database, were used as reference materials. Dual-energy signal of the liquid phantom at different breast densities (0% to 100%) and thicknesses (1 to 8 cm) were simulated. Glandular and adipose tissue thicknesses were estimated from a higher order polynomial of the signals. Our results indicated that the linear attenuation coefficients of the breast-equivalent liquid phantoms match those of the target material. Comparison between measured and known breast density data shows a linear correlation with a slope close to 1 and a non-zero intercept of 7%, while plastic phantoms showed a slope of 0.6 and a non-zero intercept of 8%. Breast density results derived from the liquid calibration phantoms showed higher accuracy than those derived from the plastic phantoms for different breast thicknesses and various tube voltages. We performed experimental phantom studies using liquid phantoms and then compared the computed breast density with those obtained using a bovine tissue model. The experimental data and the known values were in good correlation with a slope close to 1 (˜1.1). In conclusion, our results indicate that liquid phantoms are a reliable alternative for calibration in dual-energy mammography and better reproduce the chemical properties of the target material.

  4. Reach for Reference. Four Recent Reference Books

    ERIC Educational Resources Information Center

    Safford, Barbara Ripp

    2004-01-01

    This article provides descriptions of four new science and technology encyclopedias that are appropriate for inclusion in upper elementary and/or middle school reference collections. "The Macmillan Encyclopedia of Weather" (Stern, Macmillan Reference/Gale), a one-volume encyclopedia for upper elementary and middle level students, is a…

  5. Identification and quantification of metabolites of AB-CHMINACA in a urine specimen of an abuser.

    PubMed

    Wurita, Amin; Hasegawa, Koutaro; Minakata, Kayoko; Gonmori, Kunio; Nozawa, Hideki; Yamagishi, Itaru; Suzuki, Osamu; Watanabe, Kanako

    2016-03-01

    We experienced an autopsy case in which the cause of death was judged as poisoning by multiple new psychoactive substances, including AB-CHMINACA, 5-fluoro-AMB and diphenidine [Forensic Toxicol. 33 (2015): 45-53]. Although unchanged AB-CHMINACA could be detected from 8 solid tissues, it could neither be detected from blood nor urine specimens. In this article, we obtained eight kinds of reference standards of AB-CHMINACA metabolites from a commercial source. The AB-CHMINACA metabolites from the urine specimen of the abuser were extracted by a modified QuEChERS method and analyzed by liquid chromatography-tandem mass spectrometry before and after hydrolysis with β-glucuronidase. Among the eight AB-CHMINACA metabolites tested, only 2 metabolites could be identified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, the concentrations of the two metabolites were not increased, suggesting that the metabolites were not in the conjugated forms. The metabolites detected were 4-hydroxycyclohexylmethyl AB-CHMINACA (M1), followed by N-[[1-(cyclohexylmethyl)-1H-indazol-3-yl]carbonyl]-l-valine (M3). Their concentrations were 52.8 ± 3.44 and 41.3 ± 5.04 ng/ml (n=10) for M1 and M3, respectively. Although there is one preceding report showing the estimations of metabolism of AB-CHMINACA without reference standards, this is the first report dealing with exact identification using reference standards, and quantification of M1 and M3 in an authentic urine specimen.

  6. MODEL VALIDATION AND UNCERTAINTY QUANTIFICATION.

    SciTech Connect

    Hemez, F.M.; Doebling, S.W.

    2000-10-01

    This session offers an open forum to discuss issues and directions of research in the areas of model updating, predictive quality of computer simulations, model validation and uncertainty quantification. Technical presentations review the state-of-the-art in nonlinear dynamics and model validation for structural dynamics. A panel discussion introduces the discussion on technology needs, future trends and challenges ahead with an emphasis placed on soliciting participation of the audience, One of the goals is to show, through invited contributions, how other scientific communities are approaching and solving difficulties similar to those encountered in structural dynamics. The session also serves the purpose of presenting the on-going organization of technical meetings sponsored by the U.S. Department of Energy and dedicated to health monitoring, damage prognosis, model validation and uncertainty quantification in engineering applications. The session is part of the SD-2000 Forum, a forum to identify research trends, funding opportunities and to discuss the future of structural dynamics.

  7. Fundamentals of Reference

    ERIC Educational Resources Information Center

    Mulac, Carolyn M.

    2012-01-01

    The all-in-one "Reference reference" you've been waiting for, this invaluable book offers a concise introduction to reference sources and services for a variety of readers, from library staff members who are asked to work in the reference department to managers and others who wish to familiarize themselves with this important area of…

  8. Live, Digital Reference.

    ERIC Educational Resources Information Center

    Kenney, Brian

    2002-01-01

    Discusses digital reference services, also known as virtual reference, chat reference, or online reference, based on a round table discussion at the 2002 American Library Association annual conference in Atlanta. Topics include numbers and marketing; sustainability; competition and models; evaluation methods; outsourcing; staffing and training;…

  9. Statistical Reference Datasets

    National Institute of Standards and Technology Data Gateway

    Statistical Reference Datasets (Web, free access)   The Statistical Reference Datasets is also supported by the Standard Reference Data Program. The purpose of this project is to improve the accuracy of statistical software by providing reference datasets with certified computational results that enable the objective evaluation of statistical software.

  10. Uncertainty Quantification for Airfoil Icing

    NASA Astrophysics Data System (ADS)

    DeGennaro, Anthony Matteo

    Ensuring the safety of airplane flight in icing conditions is an important and active arena of research in the aerospace community. Notwithstanding the research, development, and legislation aimed at certifying airplanes for safe operation, an analysis of the effects of icing uncertainties on certification quantities of interest is generally lacking. The central objective of this thesis is to examine and analyze problems in airfoil ice accretion from the standpoint of uncertainty quantification. We focus on three distinct areas: user-informed, data-driven, and computational uncertainty quantification. In the user-informed approach to uncertainty quantification, we discuss important canonical icing classifications and show how these categories can be modeled using a few shape parameters. We then investigate the statistical effects of these parameters. In the data-driven approach, we build statistical models of airfoil ice shapes from databases of actual ice shapes, and quantify the effects of these parameters. Finally, in the computational approach, we investigate the effects of uncertainty in the physics of the ice accretion process, by perturbing the input to an in-house numerical ice accretion code that we develop in this thesis.

  11. Quantification of Ceroid and Lipofuscin in Skeletal Muscle

    PubMed Central

    Tohma, Hatice; Hepworth, Anna R.; Shavlakadze, Thea; Grounds, Miranda D.; Arthur, Peter G.

    2011-01-01

    Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin). PMID:21804079

  12. High-performance liquid chromatography profiling of the major carotenoids in Arabidopsis thaliana leaf tissue.

    PubMed

    Taylor, Kerry L; Brackenridge, Anika E; Vivier, Melané A; Oberholster, Anita

    2006-07-14

    Carotenoids are extremely sensitive to a variety of physico-chemical attacks which may have a profound effect on their characteristic properties, thereby influencing the accurate identification and quantification of individual compounds. In this light, a comprehensive summary of the pitfalls encountered and precautions to be administered during handling and storage of authentic standards and samples was found to be incomplete. Furthermore, acceptable baseline separation of trans-lutein from trans-zeaxanthin and between the cis- and trans-forms of neoxanthin and violaxanthin has not been satisfactorily demonstrated. Hence the most optimal sample preparation and analytical steps were determined and a sensitive and reproducible method for the quantitative HPLC profiling of the principal carotenoids found in plant leaf tissue was developed. A reverse-phase C(30) column with a binary mobile solvent system was used for the baseline separation of eight of the major carotenoids and the two chlorophylls (a and b) within 18min. These compounds were identified via the use of authentic standards, their spectral characteristics and HPLC-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) confirmation. This method has been successfully applied for the quantification of plant pigments in Arabidopsis thaliana wild-type (WT) leaf tissue and in two A. thaliana non-photochemical mutants, namely npq1 and npq2. These mutants have previously been well-characterised and provided valuable reference data as well as acting as internal controls for the assessment of our new method.

  13. Reference genes for quantitative gene expression studies in multiple avian species.

    PubMed

    Olias, Philipp; Adam, Iris; Meyer, Anne; Scharff, Constance; Gruber, Achim D

    2014-01-01

    Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

  14. Quantification of Lung Metastases from In Vivo Mouse Models.

    PubMed

    Chang, Joan; Erler, Janine T

    2016-01-01

    Cancer research has made significant progress in terms of understanding and targeting primary tumors; however, the challenge remains for the successful treatment of metastatic cancers. This highlights the importance to use in vivo models to study the metastatic process, as well as for preclinical testing of compounds that could inhibit metastasis. As a result, proper quantification of metastases from in vivo models is of the utmost significance. Here, we provide a detailed protocol for collecting and handling lung tissues from mice, and guidance for subsequent analysis of metastases, as well as interpretation of data.

  15. Nuclear magnetic resonance-based quantification of organic diphosphates.

    PubMed

    Lenevich, Stepan; Distefano, Mark D

    2011-01-15

    Phosphorylated compounds are ubiquitous in life. Given their central role, many such substrates and analogs have been prepared for subsequent evaluation. Prior to biological experiments, it is typically necessary to determine the concentration of the target molecule in solution. Here we describe a method where concentrations of stock solutions of organic diphosphates and bisphosphonates are quantified using (31)P nuclear magnetic resonance (NMR) spectroscopy with standard instrumentation using a capillary tube with a secondary standard. The method is specific and is applicable down to a concentration of 200 μM. The capillary tube provides the reference peak for quantification and deuterated solvent for locking.

  16. Radio-frequency energy quantification in magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Alon, Leeor

    Mapping of radio frequency (RF) energy deposition has been challenging for 50+ years, especially, when scanning patients in the magnetic resonance imaging (MRI) environment. As result, electromagnetic simulation software is often used for estimating the specific absorption rate (SAR), the rate of RF energy deposition in tissue. The thesis work presents challenges associated with aligning information provided by electromagnetic simulation and MRI experiments. As result of the limitations of simulations, experimental methods for the quantification of SAR were established. A system for quantification of the total RF energy deposition was developed for parallel transmit MRI (a system that uses multiple antennas to excite and image the body). The system is capable of monitoring and predicting channel-by-channel RF energy deposition, whole body SAR and capable of tracking potential hardware failures that occur in the transmit chain and may cause the deposition of excessive energy into patients. Similarly, we demonstrated that local RF power deposition can be mapped and predicted for parallel transmit systems based on a series of MRI temperature mapping acquisitions. Resulting from the work, we developed tools for optimal reconstruction temperature maps from MRI acquisitions. The tools developed for temperature mapping paved the way for utilizing MRI as a diagnostic tool for evaluation of RF/microwave emitting device safety. Quantification of the RF energy was demonstrated for both MRI compatible and non-MRI-compatible devices (such as cell phones), while having the advantage of being noninvasive, of providing millimeter resolution and high accuracy.

  17. Quantification of Lung PET Images: Challenges and Opportunities.

    PubMed

    Chen, Delphine L; Cheriyan, Joseph; Chilvers, Edwin R; Choudhury, Gourab; Coello, Christopher; Connell, Martin; Fisk, Marie; Groves, Ashley M; Gunn, Roger N; Holman, Beverley F; Hutton, Brian F; Lee, Sarah; MacNee, William; Mohan, Divya; Parr, David; Subramanian, Deepak; Tal-Singer, Ruth; Thielemans, Kris; van Beek, Edwin J R; Vass, Laurence; Wellen, Jeremy W; Wilkinson, Ian; Wilson, Frederick J

    2017-02-01

    Millions of people are affected by respiratory diseases, leading to a significant health burden globally. Because of the current insufficient knowledge of the underlying mechanisms that lead to the development and progression of respiratory diseases, treatment options remain limited. To overcome this limitation and understand the associated molecular changes, noninvasive imaging techniques such as PET and SPECT have been explored for biomarker development, with (18)F-FDG PET imaging being the most studied. The quantification of pulmonary molecular imaging data remains challenging because of variations in tissue, air, blood, and water fractions within the lungs. The proportions of these components further differ depending on the lung disease. Therefore, different quantification approaches have been proposed to address these variabilities. However, no standardized approach has been developed to date. This article reviews the data evaluating (18)F-FDG PET quantification approaches in lung diseases, focusing on methods to account for variations in lung components and the interpretation of the derived parameters. The diseases reviewed include acute respiratory distress syndrome, chronic obstructive pulmonary disease, and interstitial lung diseases such as idiopathic pulmonary fibrosis. Based on review of prior literature, ongoing research, and discussions among the authors, suggested considerations are presented to assist with the interpretation of the derived parameters from these approaches and the design of future studies.

  18. Quantification of the vocal folds’ dynamic displacements

    NASA Astrophysics Data System (ADS)

    del Socorro Hernández-Montes, María; Muñoz, Silvino; De La Torre, Manuel; Flores, Mauricio; Pérez, Carlos; Mendoza-Santoyo, Fernando

    2016-05-01

    Fast dynamic data acquisition techniques are required to investigate the motional behavior of the vocal folds (VFs) when they are subjected to a steady air-flow through the trachea. High-speed digital holographic interferometry (DHI) is a non-invasive full-field-of-view technique that has proved its usefulness to study rapid and non-repetitive object movements. Hence it is an ideal technique used here to measure VF displacements and vibration patterns at 2000 fps. Analyses from a set of 200 displacement images showed that VFs’ vibration cycles are established along their width (y) and length (x). Furthermore, the maximum deformation for the right and left VFs’ area may be quantified from these images, which in itself represents an important result in the characterization of this structure. At a controlled air pressure, VF displacements fall within the range ~100-1740 nm, with a calculated precision and accuracy that yields a variation coefficient of 1.91%. High-speed acquisition of full-field images of VFs and their displacement quantification are on their own significant data in the study of their functional and physiological behavior since voice quality and production depend on how they vibrate, i.e. their displacement amplitude and frequency. Additionally, the use of high speed DHI avoids prolonged examinations and represents a significant scientific and technological alternative contribution in advancing the knowledge and working mechanisms of these tissues.

  19. Genetics Home Reference: cherubism

    MedlinePlus

    ... of old bone tissue with new bone (bone remodeling) and certain immune system cells. Mutations in the ... cells that break down bone tissue during bone remodeling. An excess of these bone-eating cells contributes ...

  20. Genetics Home Reference: hypochondrogenesis

    MedlinePlus

    ... for the normal development of bones and other connective tissues that form the body's supportive framework. Mutations in ... II collagen molecules, which prevents bones and other connective tissues from developing properly. Learn more about the gene ...

  1. Genetics Home Reference: achondrogenesis

    MedlinePlus

    ... for the normal development of bones and other connective tissues that form the body's supportive framework. Mutations in ... II collagen molecules, which prevents bones and other connective tissues from developing properly. Learn more about the genes ...

  2. Genetics Home Reference: fibrochondrogenesis

    MedlinePlus

    ... flexible tissue that makes up much of the skeleton during early development. Most cartilage is later converted ... tissues, impairing the formation of bones throughout the skeleton and causing changes in the eye and inner ...

  3. Genetics Home Reference

    MedlinePlus

    Skip Navigation Bar Home Current Issue Past Issues Genetics Home Reference Past Issues / Spring 2007 Table of ... of this page please turn Javascript on. The Genetics Home Reference (GHR) Web site — ghr.nlm.nih. ...

  4. Hemispheric specialization in quantification processes.

    PubMed

    Pasini, M; Tessari, A

    2001-01-01

    Three experiments were carried out to study hemispheric specialization for subitizing (the rapid enumeration of small patterns) and counting (the serial quantification process based on some formal principles). The experiments consist of numerosity identification of dot patterns presented in one visual field, with a tachistoscopic technique, or eye movements monitored through glasses, and comparison between centrally presented dot patterns and lateralized tachistoscopically presented digits. Our experiments show left visual field advantage in the identification and comparison tasks in the subitizing range, whereas right visual field advantage has been found in the comparison task for the counting range.

  5. Herbal reference standards.

    PubMed

    Schwarz, Michael; Klier, Bernhard; Sievers, Hartwig

    2009-06-01

    This review describes the current definitions and regulatory requirements that apply to reference standards that are used to analyse herbal products. It also describes and discusses the current use of reference substances and reference extracts in the European and United States pharmacopoeias.

  6. Academic Library Reference Services.

    ERIC Educational Resources Information Center

    Batt, Fred

    This examination of the philosophy and objectives of academic library reference services provides an overview of the major reference approaches to fulfilling the following primary objectives of reference services: (1) providing accurate answers to patrons' questions and/or helping patrons find sources to pursue their research needs; (2) building…

  7. Polarized light interaction with tissues

    NASA Astrophysics Data System (ADS)

    Tuchin, Valery V.

    2016-07-01

    This tutorial-review introduces the fundamentals of polarized light interaction with biological tissues and presents some of the recent key polarization optical methods that have made possible the quantitative studies essential for biomedical diagnostics. Tissue structures and the corresponding models showing linear and circular birefringence, dichroism, and chirality are analyzed. As the basis for a quantitative description of the interaction of polarized light with tissues, the theory of polarization transfer in a random medium is used. This theory employs the modified transfer equation for Stokes parameters to predict the polarization properties of single- and multiple-scattered optical fields. The near-order of scatterers in tissues is accounted for to provide an adequate description of tissue polarization properties. Biomedical diagnostic techniques based on polarized light detection, including polarization imaging and spectroscopy, amplitude and intensity light scattering matrix measurements, and polarization-sensitive optical coherence tomography are described. Examples of biomedical applications of these techniques for early diagnostics of cataracts, detection of precancer, and prediction of skin disease are presented. The substantial reduction of light scattering multiplicity at tissue optical clearing that leads to a lesser influence of scattering on the measured intrinsic polarization properties of the tissue and allows for more precise quantification of these properties is demonstrated.

  8. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

    PubMed Central

    Paolacci, Anna R; Tanzarella, Oronzo A; Porceddu, Enrico; Ciaffi, Mario

    2009-01-01

    Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions

  9. Managing tissue bank access to the OR for tissue recovery.

    PubMed

    Trim, Robert S

    2011-08-01

    Hospitals often have tissue recovery service agreements with regional tissue banks to facilitate the donation process. The agreements that outline the tissue bank-hospital relationship frequently allow tissue bank personnel to perform tissue recovery procedures in the referring hospital's OR and may or may not specify any preparatory orientation or any written protocols for tissue bank staff members to follow. This creates the potential for unintentional breaches of protocol that can affect operation of equipment or result in contamination that may put surgical patients and staff members at risk. The OR manager is responsible for establishing appropriate orientation plans for tissue bank employees to ensure they understand and adhere to the hospital's protocols.

  10. Quantification of structural changes in acute inflammation by fractal dimension, angular second moment and correlation.

    PubMed

    Stankovic, Marija; Pantic, Igor; De Luka, Silvio R; Puskas, Nela; Zaletel, Ivan; Milutinovic-Smiljanic, Sanja; Pantic, Senka; Trbovich, Alexander M

    2016-03-01

    The aim of the study was to examine alteration and possible application of fractal dimension, angular second moment, and correlation for quantification of structural changes in acutely inflamed tissue. Acute inflammation was induced by injection of turpentine oil into the right and left hind limb muscles of mice, whereas control animals received intramuscular saline injection. After 12 h, animals were anesthetised and treated muscles collected. The tissue was stained by hematoxylin and eosin, digital micrographs produced, enabling determination of fractal dimension of the cells, angular second moment and correlation of studied tissue. Histopathological analysis showed presence of inflammatory infiltrate and tissue damage in inflammatory group, whereas tissue structure in control group was preserved, devoid of inflammatory infiltrate. Fractal dimension of the cells, angular second moment and correlation of treated tissue in inflammatory group decreased in comparison to the control group. In this study, we were first to observe and report that fractal dimension of the cells, angular second moment, and correlation were reduced in acutely inflamed tissue, indicating loss of overall complexity of the cells in the tissue, the tissue uniformity and structure regularity. Fractal dimension, angular second moment and correlation could be useful methods for quantification of structural changes in acute inflammation.

  11. Detection and Quantification of Neurotransmitters in Dialysates

    PubMed Central

    Zapata, Agustin; Chefer, Vladimir I.; Shippenberg, Toni S.; Denoroy, Luc

    2010-01-01

    Sensitive analytical methods are needed for the separation and quantification of neurotransmitters obtained in microdialysate studies. This unit describes methods that permit quantification of nanomolar concentrations of monoamines and their metabolites (high-pressure liquid chromatography electrochemical detection), acetylcholine (HPLC-coupled to an enzyme reactor), and amino acids (HPLC-fluorescence detection; capillary electrophoresis with laser-induced fluorescence detection). PMID:19575473

  12. First derivative of NIR light diffuse reflectance spectra as an approach to analyze muscle tissue chromophores and light pathlength

    NASA Astrophysics Data System (ADS)

    Gussakovsky, Eugene

    2009-02-01

    Diffuse reflectance was applied to the biomedical studies (muscles, cardiac tissues etc.) in a form of either a direct pseudo-optical spectrum or its second derivative. The first derivative adopts advantages of both direct spectrum (high signal-to-noise ratio) and its second derivative (simplifying the consideration of light scattering contribution, S). In contrast to spectrophotometry of solutions, diffuse reflectance application to the analysis of turbid medium chromophores leads to non-trivial problems of contribution of light scattering, the choice of reference, and light pathlength. Under certain conditions, the first approximation of the Taylor series of S results in the known linear dependence of S on wavelength in the 650-1050 nm wavelength range. Then the light scattering contribution to the first derivative becomes a wavelength-independent offset. In contrast to the second derivative, the information on light scattering inside the tissue is not lost. Effect of reference on the measured spectra becomes negligible. Application of the first derivative allowed (i) determination of NIR light pathlength in muscle tissue, and (ii) quantification of hemoglobin + myoglobin absolute concentration (in mM) in cardiac tissue during open-heart surgery. The first derivative approach may in general be applied to any chromophores in turbid (biological) media.

  13. Tissue types (image)

    MedlinePlus

    There are 4 basic types of tissue: connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Connective tissue supports other tissues and binds them together (bone, blood, and lymph tissues). ...

  14. In vivo quantification of cochlin in glaucomatous DBA/2J mice using optical coherence tomography

    PubMed Central

    Wang, Jianhua; Aljohani, Ayman; Carreon, Teresia; Gregori, Giovanni; Bhattacharya, Sanjoy K.

    2015-01-01

    The expression of cochlin in the trabecular meshwork (TM) precedes the clinical glaucoma symptoms in DBA/2J mice. The ability to quantify cochlin in the local tissue (TM) offers potential diagnostic and prognostic values. We present two (spectroscopic and magnetomotive) optical coherence tomography (OCT) approaches for in vivo cochlin quantification in a periodic manner. The cochlin-antibody OCT signal remains stable for up to 24 hours as seen at 3.5 hours after injection allowing for repeated quantification in the living mouse eyes. PMID:26047051

  15. Automatic Quantification of the Number of Intracellular Compartments in Arabidopsis thaliana Root Cells

    PubMed Central

    Bayle, Vincent; Platre, Matthieu Pierre; Jaillais, Yvon

    2017-01-01

    In the era of quantitative biology, it is increasingly required to quantify confocal microscopy images. If possible, quantification should be performed in an automatic way, in order to avoid bias from the experimenter, to allow the quantification of a large number of samples, and to increase reproducibility between laboratories. In this protocol, we describe procedures for automatic counting of the number of intracellular compartments in Arabidopsis root cells, which can be used for example to study endocytosis or secretory trafficking pathways and to compare membrane organization between different genotypes or treatments. While developed for Arabidopsis roots, this method can be used on other tissues, cell types and plant species. PMID:28255574

  16. A simple dot-blot-Sirius red-based assay for collagen quantification.

    PubMed

    Rodríguez-Rodríguez, Pilar; Arribas, Silvia M; de Pablo, Angel Luis López; González, M Carmen; Abderrahim, Fatima; Condezo-Hoyos, Luis

    2013-08-01

    The assessment of collagen content in tissues is important in biomedical research, since this protein is altered in numerous diseases. Hydroxyproline and Sirius red based assays are the most common methods for collagen quantification. However, these procedures have some pitfalls, such as the requirement of oxygen-free medium or expensive equipment and large sample size or being unsuitable for hydrolyzed collagen, respectively. Our objective was to develop a specific, versatile, and user-friendly quantitative method applicable to small tissue samples and extracts obtained from elastin purification, therefore, suitable for simultaneous quantification of elastin. This method is based on the binding of Sirius red to collagen present in a sample immobilized on a PVDF membrane, as in the dot-blot technique, and quantified by a scanner and image analysis software. Sample loading, Sirius red concentration, temperature and incubation time, type of standard substance, albumin interference, and quantification time are optimized. The method enabled the quantification of (1) intact collagen in several rat tissue homogenates, including small resistance-sized arteries, (2) partially hydrolyzed collagen obtained from NaOH extracts, compatible with elastin purification, and (3) for the detection of differences in collagen content between hypertensive and normotensive rats. We conclude that the developed technique can be widely used since it is versatile (quantifies intact and hydrolyzed collagen), requires small sample volumes, is user-friendly (low-cost, easy to use, minimum toxic materials, and reduced time of test), and is specific (minimal interference with serum albumin).

  17. TEPC performance for a reference standard.

    PubMed

    Zhang, Weihua; Wang, Zhiqiang; Liu, Yina; Li, Chunjuan; Xiao, Xuefu; Luo, Hailong; Chen, Jun; Li, Wei

    2014-01-01

    A portable tissue-equivalent proportional counter (TEPC)-based system was developed, which will be used as a reference standard in the neutron mixed field for radiation protection. In this paper, microdosimetry of the TEPC system was studied both in neutron ((252)Cf and (241)Am-Be sources) and gamma ((137)Cs and (60)Co sources) reference radiation fields. The measured neutron and gamma-dose equivalent rates were, respectively, compared with those of the reference ambient dose equivalent rate H(*)(10). And the measured microdosimetric spectra were compared with the Monte Carlo code FLUKA simulated results. All of the comparisons primarily agreed well.

  18. Validation of the quantification of histamine in rat brain using the radioenzymatic assay

    SciTech Connect

    Russell, W.L.; Henry, D.P.

    1986-03-01

    Quantification of histamine (Hm) in rat brain is problematic. The authors have evaluated tissue extraction procedures and validated a new Hm radioenzymatic assay for brain. Brain was extracted twice with either 0.1M perchloric acid (PCA), boiling water, or water. Sacrifice by microwave irradiation increased whole brain Hm from 64.4 +/- 16.5 to 450.9 +/- 90.8 ng/g. The validity of the assay was demonstrated by isolating the product formed by the radioenzymatic assay and subjecting it to TLC and autoradiography. The sensitivity of this assay permitted the quantification of Hm in as little as 4.3 ..mu..g of hypothalamus. The authors conclude: 1) The preferred method for sacrifice is decapitation; 2) homogenization in PCA effectively extracts Hm from brain tissue; 3) the radioenzymatic assay is specific for Hm in this tissue.

  19. High frequency reference electrode

    DOEpatents

    Kronberg, James W.

    1994-01-01

    A high frequency reference electrode for electrochemical experiments comprises a mercury-calomel or silver-silver chloride reference electrode with a layer of platinum around it and a layer of a chemically and electrically resistant material such as TEFLON around the platinum covering all but a small ring or "halo" at the tip of the reference electrode, adjacent to the active portion of the reference electrode. The voltage output of the platinum layer, which serves as a redox electrode, and that of the reference electrode are coupled by a capacitor or a set of capacitors and the coupled output transmitted to a standard laboratory potentiostat. The platinum may be applied by thermal decomposition to the surface of the reference electrode. The electrode provides superior high-frequency response over conventional electrodes.

  20. High frequency reference electrode

    DOEpatents

    Kronberg, J.W.

    1994-05-31

    A high frequency reference electrode for electrochemical experiments comprises a mercury-calomel or silver-silver chloride reference electrode with a layer of platinum around it and a layer of a chemically and electrically resistant material such as TEFLON around the platinum covering all but a small ring or halo' at the tip of the reference electrode, adjacent to the active portion of the reference electrode. The voltage output of the platinum layer, which serves as a redox electrode, and that of the reference electrode are coupled by a capacitor or a set of capacitors and the coupled output transmitted to a standard laboratory potentiostat. The platinum may be applied by thermal decomposition to the surface of the reference electrode. The electrode provides superior high-frequency response over conventional electrodes. 4 figs.

  1. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Gentiana macrophylla

    PubMed Central

    He, Yihan; Yan, Hailing; Hua, Wenping; Huang, Yaya; Wang, Zhezhi

    2016-01-01

    Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates – five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) – using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔCt, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions. PMID:27446172

  2. Fast and Sensitive Method for Determination of Domoic Acid in Mussel Tissue

    PubMed Central

    Barbaro, Elena; Zangrando, Roberta; Barbante, Carlo; Gambaro, Andrea

    2016-01-01

    Domoic acid (DA), a neurotoxic amino acid produced by diatoms, is the main cause of amnesic shellfish poisoning (ASP). In this work, we propose a very simple and fast analytical method to determine DA in mussel tissue. The method consists of two consecutive extractions and requires no purification steps, due to a reduction of the extraction of the interfering species and the application of very sensitive and selective HILIC-MS/MS method. The procedural method was validated through the estimation of trueness, extract yield, precision, detection, and quantification limits of analytical method. The sample preparation was also evaluated through qualitative and quantitative evaluations of the matrix effect. These evaluations were conducted both on the DA-free matrix spiked with known DA concentration and on the reference certified material (RCM). We developed a very selective LC-MS/MS method with a very low value of method detection limit (9 ng g−1) without cleanup steps. PMID:26904720

  3. Preparing the references.

    PubMed

    Peh, W C G; Ng, K H

    2009-07-01

    In a scientific paper, the references serve to provide background information and allow the researcher to compare and contrast the work of others in relation to his own study. Authors are responsible for the accuracy of all references cited. The references quoted should be easily accessible and retrievable by anyone wishing to obtain further information. There is a strong preference for citing journal articles listed in PubMed. The two major reference format systems are the Vancouver and Harvard systems, with increasing preference for the Vancouver system. Authors should adhere exactly to the instructions to authors of the target journal.

  4. Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.

    PubMed

    Pavšič, Jernej; Devonshire, Alison; Blejec, Andrej; Foy, Carole A; Van Heuverswyn, Fran; Jones, Gerwyn M; Schimmel, Heinz; Žel, Jana; Huggett, Jim F; Redshaw, Nicholas; Karczmarczyk, Maria; Mozioğlu, Erkan; Akyürek, Sema; Akgöz, Müslüm; Milavec, Mojca

    2017-04-01

    Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.

  5. Renal tissue thawed for 30 minutes is still suitable for gene expression analysis.

    PubMed

    Ma, Yi; Kang, Xiao-Nan; Ding, Wen-Bin; Yang, Hao-Zheng; Wang, Ye; Zhang, Jin; Huang, Yi-Ran; Dai, Hui-Li

    2014-01-01

    Some biosamples obtained from biobanks may go through thawing before processing. We aim to evaluate the effects of thawing at room temperature for different time periods on gene expression analysis. A time course study with four time points was conducted to investigate the expression profiling on 10 thawed normal mice renal tissue samples through Affymetrix GeneChip mouse gene 2.0 st array. Microarray results were validated by quantitative real time polymerase chain reactions (qPCR) on 6 candidate reference genes and 11 target genes. Additionally, we used geNorm plus and NormFinder to identify the most stably expressed reference genes over time. The results showed RNA degraded more after longer incubation at room temperature. However, microarray results showed only 240 genes (0.91%) altered significantly in response to thawing at room temperature. The signal of majority altered probe sets decreased with thawing time, and the crossing point (Cp) values of all candidate reference genes correlated positively with the thawing time (p<0.05). The combination of B2M, ACTB and PPIA was identified as the best choice for qPCR normalization. We found most target genes were stable by using this normalization method. However, serious gene quantification errors were resulted from improper reference genes. In conclusion, thirty minutes of thawing at room temperature has a limited impact on microarray and qPCR analysis, gene expression variations due to RNA degradation in early period after thawing can be largely reduced by proper normalization.

  6. Determining Steady-state Tissue Residues for Invertebrates in Contaminated Sediment

    DTIC Science & Technology

    2010-05-01

    involves quantification of compounds in tissues via laboratory bioaccumulation exposures of benthic invertebrates. However, the standard 28-day expo...Harbor to assess bioaccumulation of PAHs, PCBs, chlor- inated pesticides, dioxins, and Hg by sampling tissue during seven suc- cessive time points...day tissue resi- dues may underestimate bioaccumulation and subsequently risk to benthos and higher trophic level organisms. The results of this

  7. Selection of endogenous reference microRNA genes for quantitative reverse transcription polymerase chain reaction studies of boar spermatozoa cryopreservation.

    PubMed

    Zhang, Yan; Zeng, Chang-Jun; He, Lian; Ding, Li; Tang, Ke-Yi; Peng, Wen-Pei

    2015-03-01

    It is important to select high-quality reference genes for the accurate interpretation of quantitative reverse transcription polymerase chain reaction data, in particular for certain miRNAs that may demonstrate unstable expression. Although several studies have attempted to validate reference miRNA genes in the porcine testis, spermatozoa, and other tissues, no validation studies have been carried out on cryopreserved boar spermatozoa. In this study, 15 commonly used reference miRNA genes (5S, let-7c-5p, ssc-miR-16-5p, ssc-miR-17-5p, ssc-miR-20a, ssc-miR-23a, ssc-miR-24-3p, ssc-miR-26a, ssc-miR-27a-3p, ssc-miR-92a, ssc-miR-103-3p, ssc-miR-106a, ssc-miR-107-3p, ssc-miR-186, and ssc-miR-221-3p) were selected to evaluate the expression stability of target miRNAs in boar spermatozoa under different experimental conditions and concentrations. The stability of the expression of these reference miRNAs across each sample was evaluated using geNorm, NormFinder, and BestKeeper software. The results showed that ssc-miR-186 (mean rank value = 5.00), ssc-miR-23a (5.33), and ssc-miR-27a (5.33) were the most suitable reference genes using three different statistical algorithms and comprehensive ranking. The identification of these reference miRNAs will allow for more accurate quantification of the changes in miRNA expression during cryopreservation of boar spermatozoa.

  8. Evaluation and Selection of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Withania somnifera (L.) Dunal

    PubMed Central

    Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar

    2015-01-01

    Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

  9. Processing and domain selection: Quantificational variability effects

    PubMed Central

    Harris, Jesse A.; Clifton, Charles; Frazier, Lyn

    2014-01-01

    Three studies investigated how readers interpret sentences with variable quantificational domains, e.g., The army was mostly in the capital, where mostly may quantify over individuals or parts (Most of the army was in the capital) or over times (The army was in the capital most of the time). It is proposed that a general conceptual economy principle, No Extra Times (Majewski 2006, in preparation), discourages the postulation of potentially unnecessary times, and thus favors the interpretation quantifying over parts. Disambiguating an ambiguously quantified sentence to a quantification over times interpretation was rated as less natural than disambiguating it to a quantification over parts interpretation (Experiment 1). In an interpretation questionnaire, sentences with similar quantificational variability were constructed so that both interpretations of the sentence would require postulating multiple times; this resulted in the elimination of the preference for a quantification over parts interpretation, suggesting the parts preference observed in Experiment 1 is not reducible to a lexical bias of the adverb mostly (Experiment 2). An eye movement recording study showed that, in the absence of prior evidence for multiple times, readers exhibit greater difficulty when reading material that forces a quantification over times interpretation than when reading material that allows a quantification over parts interpretation (Experiment 3). These experiments contribute to understanding readers’ default assumptions about the temporal properties of sentences, which is essential for understanding the selection of a domain for adverbial quantifiers and, more generally, for understanding how situational constraints influence sentence processing. PMID:25328262

  10. "Absolute" quantification in magnetic resonance spectroscopy: validation of a clinical protocol in multiple sclerosis.

    PubMed

    Bagory, Matthieu; Durand-Dubief, Françoise; Ibarrola, Danielle; Confavreux, Christian; Sappey-Marinier, Dominique

    2007-01-01

    MRS allows to measure cerebral metabolites, thus helping to characterize brain disease diagnosis and followup. Metabolite concentration quantification is usually based on metabolite ratio referring to creatine. If this metabolite concentration is supposed to be constant, it may vary in pathological processes. Therefore, "absolute" concentration methodology is needed. The aim of this study is to validate a clinical "absolute" quantification protocol through the development of an external metabolic phantom, calibration and correction, and the investigation of reproducibility issues. When phantom stability was investigated by a short-term and a long-term reproducibility study, both Standard Deviations (SD) were in agreement with literature values. This "absolute" quantification method was applied to patients with Multiple Sclerosis (MS). The results show a significant decrease in both N-Acetyl Aspartate (NAA) and choline concentrations.

  11. Reference Point Heterogeneity

    PubMed Central

    Terzi, Ayse; Koedijk, Kees; Noussair, Charles N.; Pownall, Rachel

    2016-01-01

    It is well-established that, when confronted with a decision to be taken under risk, individuals use reference payoff levels as important inputs. The purpose of this paper is to study which reference points characterize decisions in a setting in which there are several plausible reference levels of payoff. We report an experiment, in which we investigate which of four potential reference points: (1) a population average payoff level, (2) the announced expected payoff of peers in a similar decision situation, (3) a historical average level of earnings that others have received in the same task, and (4) an announced anticipated individual payoff level, best describes decisions in a decontextualized risky decision making task. We find heterogeneity among individuals in the reference points they employ. The population average payoff level is the modal reference point, followed by experimenter's stated expectation of a participant's individual earnings, followed in turn by the average earnings of other participants in previous sessions of the same experiment. A sizeable share of individuals show multiple reference points simultaneously. The reference point that best fits the choices of the individual is not affected by a shock to her income. PMID:27672374

  12. Marketing Reference Services.

    ERIC Educational Resources Information Center

    Norman, O. Gene

    1995-01-01

    Relates the marketing concept to library reference services. Highlights include a review of the literature and an overview of marketing, including research, the marketing mix, strategic plan, marketing plan, and marketing audit. Marketing principles are applied to reference services through the marketing mix elements of product, price, place, and…

  13. An Online Reference System.

    ERIC Educational Resources Information Center

    Chisman, Janet; Treat, William

    1984-01-01

    Describes a computer aid developed to assist in academic library reference service using the DataPhase Circulation System, an automated system that features full cataloging records in database and permits local programing. Access points (subject, type of reference work, course) and database structure and user screens are highlighted. (EJS)

  14. China Connections Reference Book.

    ERIC Educational Resources Information Center

    Kalat, Marie B.; Hoermann, Elizabeth F.

    This reference book focuses on six aspects of the geography of the People's Republic of China. They are: territory, governing units, population and land use, waterways, land forms, and climates. Designed as a primary reference, the book explains how the Chinese people and their lifestyles are affected by China's geography. Special components…

  15. Rethinking Virtual Reference

    ERIC Educational Resources Information Center

    Tenopir, Carol

    2004-01-01

    Virtual reference services seem a natural extension of libraries digital collections and the emphasis on access to the library anytime, anywhere. If patrons use the library from home, it makes sense to provide them with person-to-person online reference. The Library of Congress (LC), OCLC, and several large library systems have developed and…

  16. Reference Point Heterogeneity.

    PubMed

    Terzi, Ayse; Koedijk, Kees; Noussair, Charles N; Pownall, Rachel

    2016-01-01

    It is well-established that, when confronted with a decision to be taken under risk, individuals use reference payoff levels as important inputs. The purpose of this paper is to study which reference points characterize decisions in a setting in which there are several plausible reference levels of payoff. We report an experiment, in which we investigate which of four potential reference points: (1) a population average payoff level, (2) the announced expected payoff of peers in a similar decision situation, (3) a historical average level of earnings that others have received in the same task, and (4) an announced anticipated individual payoff level, best describes decisions in a decontextualized risky decision making task. We find heterogeneity among individuals in the reference points they employ. The population average payoff level is the modal reference point, followed by experimenter's stated expectation of a participant's individual earnings, followed in turn by the average earnings of other participants in previous sessions of the same experiment. A sizeable share of individuals show multiple reference points simultaneously. The reference point that best fits the choices of the individual is not affected by a shock to her income.

  17. Protein inference: A protein quantification perspective.

    PubMed

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/.

  18. Integrative analysis of 111 reference human epigenomes.

    PubMed

    Kundaje, Anshul; Meuleman, Wouter; Ernst, Jason; Bilenky, Misha; Yen, Angela; Heravi-Moussavi, Alireza; Kheradpour, Pouya; Zhang, Zhizhuo; Wang, Jianrong; Ziller, Michael J; Amin, Viren; Whitaker, John W; Schultz, Matthew D; Ward, Lucas D; Sarkar, Abhishek; Quon, Gerald; Sandstrom, Richard S; Eaton, Matthew L; Wu, Yi-Chieh; Pfenning, Andreas R; Wang, Xinchen; Claussnitzer, Melina; Liu, Yaping; Coarfa, Cristian; Harris, R Alan; Shoresh, Noam; Epstein, Charles B; Gjoneska, Elizabeta; Leung, Danny; Xie, Wei; Hawkins, R David; Lister, Ryan; Hong, Chibo; Gascard, Philippe; Mungall, Andrew J; Moore, Richard; Chuah, Eric; Tam, Angela; Canfield, Theresa K; Hansen, R Scott; Kaul, Rajinder; Sabo, Peter J; Bansal, Mukul S; Carles, Annaick; Dixon, Jesse R; Farh, Kai-How; Feizi, Soheil; Karlic, Rosa; Kim, Ah-Ram; Kulkarni, Ashwinikumar; Li, Daofeng; Lowdon, Rebecca; Elliott, GiNell; Mercer, Tim R; Neph, Shane J; Onuchic, Vitor; Polak, Paz; Rajagopal, Nisha; Ray, Pradipta; Sallari, Richard C; Siebenthall, Kyle T; Sinnott-Armstrong, Nicholas A; Stevens, Michael; Thurman, Robert E; Wu, Jie; Zhang, Bo; Zhou, Xin; Beaudet, Arthur E; Boyer, Laurie A; De Jager, Philip L; Farnham, Peggy J; Fisher, Susan J; Haussler, David; Jones, Steven J M; Li, Wei; Marra, Marco A; McManus, Michael T; Sunyaev, Shamil; Thomson, James A; Tlsty, Thea D; Tsai, Li-Huei; Wang, Wei; Waterland, Robert A; Zhang, Michael Q; Chadwick, Lisa H; Bernstein, Bradley E; Costello, Joseph F; Ecker, Joseph R; Hirst, Martin; Meissner, Alexander; Milosavljevic, Aleksandar; Ren, Bing; Stamatoyannopoulos, John A; Wang, Ting; Kellis, Manolis

    2015-02-19

    The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

  19. Tissue repair

    PubMed Central

    2010-01-01

    As living beings that encounter every kind of traumatic event from paper cut to myocardial infarction, we must possess ways to heal damaged tissues. While some animals are able to regrow complete body parts following injury (such as the earthworm who grows a new head following bisection), humans are sadly incapable of such feats. Our means of recovery following tissue damage consists largely of repair rather than pure regeneration. Thousands of times in our lives, a meticulously scripted but unseen wound healing drama plays, with cells serving as actors, extracellular matrix as the setting and growth factors as the means of communication. This article briefly reviews the cells involved in tissue repair, their signaling and proliferation mechanisms and the function of the extracellular matrix, then presents the actors and script for the three acts of the tissue repair drama. PMID:21220961

  20. Tissue Issues

    ERIC Educational Resources Information Center

    Metz, James

    2016-01-01

    Every day, 27,000 trees are used to make bathroom tissue. Americans use an average of 23.6 rolls per person per year, and more than 7 billion rolls of toilet paper are sold yearly in the United States alone. Perhaps the amount of bathroom tissue used can be reduced by changing the dimensions of the paper or the core. This brief article presents…

  1. Sulfur-based absolute quantification of proteins using isotope dilution inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Lee, Hyun-Seok; Heun Kim, Sook; Jeong, Ji-Seon; Lee, Yong-Moon; Yim, Yong-Hyeon

    2015-10-01

    An element-based reductive approach provides an effective means of realizing International System of Units (SI) traceability for high-purity biological standards. Here, we develop an absolute protein quantification method using double isotope dilution (ID) inductively coupled plasma mass spectrometry (ICP-MS) combined with microwave-assisted acid digestion for the first time. We validated the method and applied it to certify the candidate protein certified reference material (CRM) of human growth hormone (hGH). The concentration of hGH was determined by analysing the total amount of sulfur in hGH. Next, the size-exclusion chromatography method was used with ICP-MS to characterize and quantify sulfur-containing impurities. By subtracting the contribution of sulfur-containing impurities from the total sulfur content in the hGH CRM, we obtained a SI-traceable certification value. The quantification result obtained with the present method based on sulfur analysis was in excellent agreement with the result determined via a well-established protein quantification method based on amino acid analysis using conventional acid hydrolysis combined with an ID liquid chromatography-tandem mass spectrometry. The element-based protein quantification method developed here can be generally used for SI-traceable absolute quantification of proteins, especially pure-protein standards.

  2. Design and characterization of a direct ELISA for the detection and quantification of leucomalachite green.

    PubMed

    Singh, Gurmit; Koerner, Terence; Gelinas, Jean-Marc; Abbott, Michael; Brady, Beth; Huet, Anne-Catherine; Charlier, Caroline; Delahaut, Philippe; Godefroy, Samuel Benrejeb

    2011-06-01

    Malachite green (MG), a member of the N-methylated triphenylmethane class of dyes, has long been used to control fungal and protozoan infections in fish. MG is easily absorbed by fish during waterborne exposure and is rapidly metabolized into leucomalachite green (LMG), which is known for its long residence time in edible fish tissue. This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of LMG in fish tissue. This development includes a simple and versatile method for the conversion of LMG to monodesmethyl-LMG, which is then conjugated to bovine serum albumin (BSA) to produce an immunogenic material. Rabbit polyclonal antibodies are generated against this immunogen, purified and used to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the screening and quantification of LMG in fish tissue. The assay performed well, with a limit of detection (LOD) and limit of quantification (LOQ) of 0.1 and 0.3 ng g(-1) of fish tissue, respectively. The average extraction efficiency from a matrix of tilapia fillets was approximately 73% and the day-to-day reproducibility for these extractions in the assay was between 5 and 10%.

  3. Design and characterization of a direct ELISA for the detection and quantification of leucomalachite green

    PubMed Central

    Singh, Gurmit; Koerner, Terence; Gelinas, Jean-Marc; Abbott, Michael; Brady, Beth; Huet, Anne-Catherine; Charlier, Caroline; Delahaut, Philippe; Godefroy, Samuel Benrejeb

    2011-01-01

    Malachite green (MG), a member of the N-methylated triphenylmethane class of dyes, has long been used to control fungal and protozoan infections in fish. MG is easily absorbed by fish during waterborne exposure and is rapidly metabolized into leucomalachite green (LMG), which is known for its long residence time in edible fish tissue. This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of LMG in fish tissue. This development includes a simple and versatile method for the conversion of LMG to monodesmethyl-LMG, which is then conjugated to bovine serum albumin (BSA) to produce an immunogenic material. Rabbit polyclonal antibodies are generated against this immunogen, purified and used to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the screening and quantification of LMG in fish tissue. The assay performed well, with a limit of detection (LOD) and limit of quantification (LOQ) of 0.1 and 0.3 ng g−1 of fish tissue, respectively. The average extraction efficiency from a matrix of tilapia fillets was approximately 73% and the day-to-day reproducibility for these extractions in the assay was between 5 and 10%. PMID:21623496

  4. Quantification of light attenuation in optically cleared mouse brains

    PubMed Central

    d’Esposito, Angela; Nikitichev, Daniil; Desjardins, Adrien; Walker-Samuel, Simon; Lythgoe, Mark F.

    2015-01-01

    Optical clearing, in combination with recently developed optical imaging techniques, enables visualization and acquisition of high resolution, three-dimensional images of biological structures deep within tissue. Many different approaches can be used to reduce light absorption and scattering within the tissue, but there is a paucity of research on the quantification of clearing efficacy. With the use of a custom-made spectroscopy system, we developed a way to quantify the quality of clearing in biological tissue, and applied it to the mouse brain. Three clearing techniques were compared: BABB (Murray’s clear), pBABB (a modification of BABB which includes the use of hydrogen peroxide) and passive CLARITY. Despite being limited to autofluorescence studies, we found that pBABB produced the highest degree of optical clearing. Furthermore, the approach allows regional measurement of light attenuation to be performed, and. our results show that light is most attenuated in regions with high lipid content. This work provides a way to choose between the multiple clearing protocols available, and it could prove useful for evaluating images that are acquired with cleared tissues. PMID:26277988

  5. Quantification of Daily Physical Activity

    NASA Technical Reports Server (NTRS)

    Whalen, Robert; Breit, Greg; Quintana, Jason

    1994-01-01

    The influence of physical activity on the maintenance and adaptation of musculoskeletal tissue is difficult to assess. Cumulative musculoskeletal loading is hard to quantify and the attributes of the daily tissue loading history affecting bone metabolism have not been completely identified. By monitoring the vertical component of the daily ground reaction force (GRFz), we have an indirect measure of cumulative daily lower limb musculoskeletal loading to correlate with bone density and structure. The objective of this research is to develop instrumentation and methods of analysis to quantify activity level in terms of the daily history of ground reaction forces.

  6. The NASA Langley Multidisciplinary Uncertainty Quantification Challenge

    NASA Technical Reports Server (NTRS)

    Crespo, Luis G.; Kenny, Sean P.; Giesy, Daniel P.

    2014-01-01

    This paper presents the formulation of an uncertainty quantification challenge problem consisting of five subproblems. These problems focus on key aspects of uncertainty characterization, sensitivity analysis, uncertainty propagation, extreme-case analysis, and robust design.

  7. Direct qPCR quantification using the Quantifiler(®) Trio DNA quantification kit.

    PubMed

    Liu, Jason Yingjie

    2014-11-01

    The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler(®) Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both low-level touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler(®) Trio kit enabled the detection of less than 10pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler(®) Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler(®) Trio kit) and the direct STR results (GlobalFiler™ PCR amplification kit(*)) for low-level touch DNA samples indicates that direct quantification using the Quantifiler(®) Trio DNA quantification kit is more reliable than the Quantifiler(®) Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10pg of DNA.

  8. Validation of reference transcripts in strawberry (Fragaria spp.).

    PubMed

    Clancy, Maureen A; Rosli, Hernan G; Chamala, Srikar; Barbazuk, W Brad; Civello, P Marcos; Folta, Kevin M

    2013-12-01

    Contemporary methods to assay gene expression depend on a stable set of reference transcripts for accurate quantitation. A lack of well-tested reference genes slows progress in characterizing gene expression in high-value specialty crops. In this study, a set of strawberry (Fragaria spp.) constitutively expressed reference genes has been identified by merging digital gene expression data with expression profiling. Constitutive reference candidates were validated using quantitative PCR and hybridization. Several transcripts have been identified that show improved stability across tissues relative to traditional reference transcripts. Results are similar between commercial octoploid strawberry and the diploid model. Our findings also show that while some never-before-used references are appropriate for most applications, even the most stable reference transcripts require careful assessment across the diverse tissues and fruit developmental states before being adopted as controls.

  9. Genetics Home Reference: sitosterolemia

    MedlinePlus

    ... with sitosterolemia , accumulation of fatty deposits in arteries ( atherosclerosis ) can occur as early as childhood. These deposits ... the arteries, skin, and other tissues, resulting in atherosclerosis, xanthomas, and the additional signs and symptoms of ...

  10. Genetics Home Reference: piebaldism

    MedlinePlus

    ... SLUG ) provides instructions for making a protein called snail 2. Research indicates that the snail 2 protein is required during embryonic growth for ... many tissues and cell types, including melanocytes. The snail 2 protein probably plays a role in the ...

  11. MAMA Software Features: Visual Examples of Quantification

    SciTech Connect

    Ruggiero, Christy E.; Porter, Reid B.

    2014-05-20

    This document shows examples of the results from quantifying objects of certain sizes and types in the software. It is intended to give users a better feel for some of the quantification calculations, and, more importantly, to help users understand the challenges with using a small set of ‘shape’ quantification calculations for objects that can vary widely in shapes and features. We will add more examples to this in the coming year.

  12. Genetics Home Reference: nephronophthisis

    MedlinePlus

    ... these are often referred to as nephronophthisis -associated ciliopathies. For example, Senior-Løken syndrome is characterized by ... Nephronophthisis Patient Support and Advocacy Resources (2 links) Ciliopathy Alliance National Kidney Foundation GeneReviews (1 link) Nephronophthisis ...

  13. Value of Information References

    SciTech Connect

    Morency, Christina

    2014-12-12

    This file contains a list of relevant references on value of information (VOI) in RIS format. VOI provides a quantitative analysis to evaluate the outcome of the combined technologies (seismology, hydrology, geodesy) used to monitor Brady's Geothermal Field.

  14. EPA QUICK REFERENCE GUIDES

    EPA Science Inventory

    EPA Quick Reference Guides are compilations of information on chemical and biological terrorist agents. The information is presented in consistent format and includes agent characteristics, release scenarios, health and safety data, real-time field detection, effect levels, samp...

  15. Selecting a reference object.

    PubMed

    Miller, Jared E; Carlson, Laura A; Hill, Patrick L

    2011-07-01

    One way to describe the location of an object is to relate it to another object. Often there are many nearby objects, each of which could serve as a candidate to be the reference object. A common theoretical assumption is that features that make a given object salient relative to the candidate set are instrumental in determining which is selected. The current research tests this assumption, assessing the relative importance of spatial, perceptual, and functional-interactive features. Three experiments demonstrated that spatial features have the strongest influence on reference object selection, with the perceptual feature of color playing no significant role. Functional-interactive features were shown to be spatially dependent, having an influence only when the spatial configuration enabled an interaction between the located object and the reference object. These findings challenge the common perspective that salience in and of itself dictates reference object selection and argue for a reliance on spatial features.

  16. Genetics Home Reference

    MedlinePlus

    ... MENU Toggle navigation Home Page Search Share: Email Facebook Twitter Home Health Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Genetics Home Reference provides consumer-friendly information about the effects of genetic variation ...

  17. Enterprise Reference Library

    NASA Technical Reports Server (NTRS)

    Bickham, Grandin; Saile, Lynn; Havelka, Jacque; Fitts, Mary

    2011-01-01

    Introduction: Johnson Space Center (JSC) offers two extensive libraries that contain journals, research literature and electronic resources. Searching capabilities are available to those individuals residing onsite or through a librarian s search. Many individuals have rich collections of references, but no mechanisms to share reference libraries across researchers, projects, or directorates exist. Likewise, information regarding which references are provided to which individuals is not available, resulting in duplicate requests, redundant labor costs and associated copying fees. In addition, this tends to limit collaboration between colleagues and promotes the establishment of individual, unshared silos of information The Integrated Medical Model (IMM) team has utilized a centralized reference management tool during the development, test, and operational phases of this project. The Enterprise Reference Library project expands the capabilities developed for IMM to address the above issues and enhance collaboration across JSC. Method: After significant market analysis for a multi-user reference management tool, no available commercial tool was found to meet this need, so a software program was built around a commercial tool, Reference Manager 12 by The Thomson Corporation. A use case approach guided the requirements development phase. The premise of the design is that individuals use their own reference management software and export to SharePoint when their library is incorporated into the Enterprise Reference Library. This results in a searchable user-specific library application. An accompanying share folder will warehouse the electronic full-text articles, which allows the global user community to access full -text articles. Discussion: An enterprise reference library solution can provide a multidisciplinary collection of full text articles. This approach improves efficiency in obtaining and storing reference material while greatly reducing labor, purchasing and

  18. Precise quantification of nanoparticle internalization.

    PubMed

    Gottstein, Claudia; Wu, Guohui; Wong, Benjamin J; Zasadzinski, Joseph Anthony

    2013-06-25

    Nanoparticles have opened new exciting avenues for both diagnostic and therapeutic applications in human disease, and targeted nanoparticles are increasingly used as specific drug delivery vehicles. The precise quantification of nanoparticle internalization is of importance to measure the impact of physical and chemical properties on the uptake of nanoparticles into target cells or into cells responsible for rapid clearance. Internalization of nanoparticles has been measured by various techniques, but comparability of data between different laboratories is impeded by lack of a generally accepted standardized assay. Furthermore, the distinction between associated and internalized particles has been a challenge for many years, although this distinction is critical for most research questions. Previously used methods to verify intracellular location are typically not quantitative and do not lend themselves to high-throughput analysis. Here, we developed a mathematical model which integrates the data from high-throughput flow cytometry measurements with data from quantitative confocal microscopy. The generic method described here will be a useful tool in biomedical nanotechnology studies. The method was then applied to measure the impact of surface coatings of vesosomes on their internalization by cells of the reticuloendothelial system (RES). RES cells are responsible for rapid clearance of nanoparticles, and the resulting fast blood clearance is one of the major challenges in biomedical applications of nanoparticles. Coating of vesosomes with long chain polyethylene glycol showed a trend for lower internalization by RES cells.

  19. Stirling Convertor Fasteners Reliability Quantification

    NASA Technical Reports Server (NTRS)

    Shah, Ashwin R.; Korovaichuk, Igor; Kovacevich, Tiodor; Schreiber, Jeffrey G.

    2006-01-01

    Onboard Radioisotope Power Systems (RPS) being developed for NASA s deep-space science and exploration missions require reliable operation for up to 14 years and beyond. Stirling power conversion is a candidate for use in an RPS because it offers a multifold increase in the conversion efficiency of heat to electric power and reduced inventory of radioactive material. Structural fasteners are responsible to maintain structural integrity of the Stirling power convertor, which is critical to ensure reliable performance during the entire mission. Design of fasteners involve variables related to the fabrication, manufacturing, behavior of fasteners and joining parts material, structural geometry of the joining components, size and spacing of fasteners, mission loads, boundary conditions, etc. These variables have inherent uncertainties, which need to be accounted for in the reliability assessment. This paper describes these uncertainties along with a methodology to quantify the reliability, and provides results of the analysis in terms of quantified reliability and sensitivity of Stirling power conversion reliability to the design variables. Quantification of the reliability includes both structural and functional aspects of the joining components. Based on the results, the paper also describes guidelines to improve the reliability and verification testing.

  20. Membrane reference electrode

    DOEpatents

    Redey, L.; Bloom, I.D.

    1988-01-21

    A reference electrode utilizes a small thin, flat membrane of a highly conductive glass placed on a small diameter insulator tube having a reference material inside in contact with an internal voltage lead. When the sensor is placed in a non-aqueous ionic electrolytic solution, the concentration difference across the glass membrane generates a low voltage signal in precise relationship to the concentration of the species to be measured, with high spatial resolution. 2 figs.

  1. Membrane reference electrode

    DOEpatents

    Redey, Laszlo; Bloom, Ira D.

    1989-01-01

    A reference electrode utilizes a small thin, flat membrane of a highly conductive glass placed on a small diameter insulator tube having a reference material inside in contact with an internal voltage lead. When the sensor is placed in a non-aqueous ionic electrolytic solution, the concentration difference across the glass membrane generates a low voltage signal in precise relationship to the concentration of the species to be measured with high spatial resolution.

  2. Precision displacement reference system

    DOEpatents

    Bieg, Lothar F.; Dubois, Robert R.; Strother, Jerry D.

    2000-02-22

    A precision displacement reference system is described, which enables real time accountability over the applied displacement feedback system to precision machine tools, positioning mechanisms, motion devices, and related operations. As independent measurements of tool location is taken by a displacement feedback system, a rotating reference disk compares feedback counts with performed motion. These measurements are compared to characterize and analyze real time mechanical and control performance during operation.

  3. Targeted proteomic quantification on quadrupole-orbitrap mass spectrometer.

    PubMed

    Gallien, Sebastien; Duriez, Elodie; Crone, Catharina; Kellmann, Markus; Moehring, Thomas; Domon, Bruno

    2012-12-01

    There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein

  4. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies

    PubMed Central

    Chapman, Joanne R.; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies. PMID:26555275

  5. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

    PubMed

    Chapman, Joanne R; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

  6. Tissue Classification

    SciTech Connect

    Robinson, David Gerald

    2015-01-01

    The project began as a e ort to support InLight and Lumidigm. With the sale of the companies to a non-New Mexico entity, the project then focused on supporting a new company Medici Technologies. The Small Business (SB) is attempting to quantify glucose in tissue using a series of short interferometer scans of the nger. Each scan is produced from a novel presentation of the nger to the device. The intent of the project is to identify and, if possible, implement improved methods for classi cation, feature selection, and training to improve the performance of predictive algorithms used for tissue classi cation.

  7. Quantification of isotopic turnover in agricultural systems

    NASA Astrophysics Data System (ADS)

    Braun, A.; Auerswald, K.; Schnyder, H.

    2012-04-01

    The isotopic turnover, which is a proxy for the metabolic rate, is gaining scientific importance. It is quantified for an increasing range of organisms, from microorganisms over plants to animals including agricultural livestock. Additionally, the isotopic turnover is analyzed on different scales, from organs to organisms to ecosystems and even to the biosphere. In particular, the quantification of the isotopic turnover of specific tissues within the same organism, e.g. organs like liver and muscle and products like milk and faeces, has brought new insights to improve understanding of nutrient cycles and fluxes, respectively. Thus, the knowledge of isotopic turnover is important in many areas, including physiology, e.g. milk synthesis, ecology, e.g. soil retention time of water, and medical science, e.g. cancer diagnosis. So far, the isotopic turnover is quantified by applying time, cost and expertise intensive tracer experiments. Usually, this comprises two isotopic equilibration periods. A first equilibration period with a constant isotopic input signal is followed by a second equilibration period with a distinct constant isotopic input signal. This yields a smooth signal change from the first to the second signal in the object under consideration. This approach reveals at least three major problems. (i) The input signals must be controlled isotopically, which is almost impossible in many realistic cases like free ranging animals. (ii) Both equilibration periods may be very long, especially when the turnover rate of the object under consideration is very slow, which aggravates the first problem. (iii) The detection of small or slow pools is improved by large isotopic signal changes, but large isotopic changes also involve a considerable change in the input material; e.g. animal studies are usually carried out as diet-switch experiments, where the diet is switched between C3 and C4 plants, since C3 and C4 plants differ strongly in their isotopic signal. The

  8. A Frequency-Shift Method to Measure Shear-Wave Attenuation in Soft Tissues.

    PubMed

    Bernard, Simon; Kazemirad, Siavash; Cloutier, Guy

    2017-03-01

    In vivo quantification of shear-wave attenuation in soft tissues may help to better understand human tissue rheology and lead to new diagnostic strategies. Attenuation is difficult to measure in acoustic radiation force elastography because the shear-wave amplitude decreases due to a combination of diffraction and viscous attenuation. Diffraction correction requires assuming a cylindrical wavefront and an isotropic propagation medium, which may not be the case in some applications. In this paper, the frequency-shift method, used in ultrasound imaging and seismology, was adapted for shear-wave attenuation measurement in elastography. This method is not sensitive to diffraction effects. For a linear frequency dependence of the attenuation, a closed-form relation was obtained between the decrease in the peak frequency of the gamma-distributed wave amplitude spectrum and the attenuation coefficient of the propagation medium. The proposed method was tested against a plane-wave reference method in homogeneous agar-gelatin phantoms with 0%, 10%, and 20% oil concentrations, and hence different attenuations of 0.117, 0.202, and 0.292 [Formula: see text]/Hz, respectively. Applicability to biological tissues was demonstrated with two ex vivo porcine liver samples (0.79 and 1.35 [Formula: see text]/Hz) and an in vivo human muscle, measured along (0.43 [Formula: see text]/Hz) and across (1.77 [Formula: see text]/Hz) the tissue fibers. In all cases, the data supported the assumptions of a gamma-distributed spectrum for the source and linear frequency attenuation for the tissue. This method provides tissue attenuation, which is relevant diagnostic information to model viscosity, in addition to shear-wave velocity used to assess elasticity. Data processing is simple and could be performed automatically in real time for clinical applications.

  9. Quantification of punctate iron sources using magnetic resonance phase.

    PubMed

    McAuley, Grant; Schrag, Matthew; Sipos, Pál; Sun, Shu-Wei; Obenaus, Andre; Neelavalli, Jaladhar; Haacke, E Mark; Holshouser, Barbara; Madácsi, Ramóna; Kirsch, Wolff

    2010-01-01

    Iron-mediated tissue damage is present in cerebrovascular and neurodegenerative diseases and neurotrauma. Brain microbleeds are often present in these maladies and are assuming increasing clinical importance. Because brain microbleeds present a source of pathologic iron to the brain, the noninvasive quantification of this iron pool is potentially valuable. Past efforts to quantify brain iron have focused on content estimation within distributed brain regions. In addition, conventional approaches using "magnitude" images have met significant limitations. In this study, a technique is presented to quantify the iron content of punctate samples using phase images. Samples are modeled as magnetic dipoles and phase shifts due to local dipole field perturbations are mathematically related to sample iron content and radius using easily recognized geometric features in phase images. Phantoms containing samples of a chitosan-ferric oxyhydroxide composite (which serves as a mimic for hemosiderin) were scanned with a susceptibility-weighted imaging sequence at 11.7 T. Plots relating sample iron content and radius to phase image features were compared to theoretical predictions. The primary result is the validation of the technique by the excellent agreement between theory and the iron content plot. This research is a potential first step toward quantification of punctate brain iron sources such as brain microbleeds.

  10. Uncertainty Quantification in Climate Modeling

    NASA Astrophysics Data System (ADS)

    Sargsyan, K.; Safta, C.; Berry, R.; Debusschere, B.; Najm, H.

    2011-12-01

    We address challenges that sensitivity analysis and uncertainty quantification methods face when dealing with complex computational models. In particular, climate models are computationally expensive and typically depend on a large number of input parameters. We consider the Community Land Model (CLM), which consists of a nested computational grid hierarchy designed to represent the spatial heterogeneity of the land surface. Each computational cell can be composed of multiple land types, and each land type can incorporate one or more sub-models describing the spatial and depth variability. Even for simulations at a regional scale, the computational cost of a single run is quite high and the number of parameters that control the model behavior is very large. Therefore, the parameter sensitivity analysis and uncertainty propagation face significant difficulties for climate models. This work employs several algorithmic avenues to address some of the challenges encountered by classical uncertainty quantification methodologies when dealing with expensive computational models, specifically focusing on the CLM as a primary application. First of all, since the available climate model predictions are extremely sparse due to the high computational cost of model runs, we adopt a Bayesian framework that effectively incorporates this lack-of-knowledge as a source of uncertainty, and produces robust predictions with quantified uncertainty even if the model runs are extremely sparse. In particular, we infer Polynomial Chaos spectral expansions that effectively encode the uncertain input-output relationship and allow efficient propagation of all sources of input uncertainties to outputs of interest. Secondly, the predictability analysis of climate models strongly suffers from the curse of dimensionality, i.e. the large number of input parameters. While single-parameter perturbation studies can be efficiently performed in a parallel fashion, the multivariate uncertainty analysis

  11. Uncertainty quantification in molecular dynamics

    NASA Astrophysics Data System (ADS)

    Rizzi, Francesco

    This dissertation focuses on uncertainty quantification (UQ) in molecular dynamics (MD) simulations. The application of UQ to molecular dynamics is motivated by the broad uncertainty characterizing MD potential functions and by the complexity of the MD setting, where even small uncertainties can be amplified to yield large uncertainties in the model predictions. Two fundamental, distinct sources of uncertainty are investigated in this work, namely parametric uncertainty and intrinsic noise. Intrinsic noise is inherently present in the MD setting, due to fluctuations originating from thermal effects. Averaging methods can be exploited to reduce the fluctuations, but due to finite sampling, this effect cannot be completely filtered, thus yielding a residual uncertainty in the MD predictions. Parametric uncertainty, on the contrary, is introduced in the form of uncertain potential parameters, geometry, and/or boundary conditions. We address the UQ problem in both its main components, namely the forward propagation, which aims at characterizing how uncertainty in model parameters affects selected observables, and the inverse problem, which involves the estimation of target model parameters based on a set of observations. The dissertation highlights the challenges arising when parametric uncertainty and intrinsic noise combine to yield non-deterministic, noisy MD predictions of target macroscale observables. Two key probabilistic UQ methods, namely Polynomial Chaos (PC) expansions and Bayesian inference, are exploited to develop a framework that enables one to isolate the impact of parametric uncertainty on the MD predictions and, at the same time, properly quantify the effect of the intrinsic noise. Systematic applications to a suite of problems of increasing complexity lead to the observation that an uncertain PC representation built via Bayesian regression is the most suitable model for the representation of uncertain MD predictions of target observables in the

  12. Multi-laboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accurate identification and quantification of Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, within root tissue and soil are important tasks. Several quantitative PCR (qPCR) assays have been developed but there are no reports comparing their use in sensitive and specific...

  13. [Muscles and connective tissue: histology].

    PubMed

    Delage, J-P

    2012-10-01

    Here, we give some comments about the DVD movies "Muscle Attitudes" from Endovivo productions, the movies up lighting some loss in the attention given to studies on the connective tissue, and especially them into muscles. The main characteristics of the different components in the intra-muscular connective tissue (perimysium, endomysium, epimysium) are shown here with special references to their ordered architecture and special references to their spatial distributions. This connective tissue is abundant into the muscles and is in continuity with the muscles in vicinity, with their tendons and their sheath, sticking the whole on skin. This connective tissue has also very abundant connections on the muscles fibres. It is then assumed that the connective tissue sticks every organs or cells of the locomotion system. Considering the elastic properties of the collagen fibres which are the most abundant component of connective tissue, it is possible to up light a panel of connective tissue associated functions such as the transmission of muscle contractions or the regulation of protein and energetic muscles metabolism.

  14. Quantification-based mass spectrometry imaging of proteins by parafilm assisted microdissection.

    PubMed

    Franck, Julien; Quanico, Jusal; Wisztorski, Maxence; Day, Robert; Salzet, Michel; Fournier, Isabelle

    2013-09-03

    MALDI mass spectrometry imaging (MALDI-MSI) was presented as a good strategy to highlight regions presenting specific phenotypes based on molecular content. The proteins present in the different areas can be identified by MALDI MSI; however, the number of protein identifications remains low in comparison with classical MS-based proteomics approaches. To overcome this, a new strategy, involving the microdissection of tissue sections mounted on parafilm M-covered glass slides, is presented. Extraction and fractionation of proteins from a specific region of interest were investigated, leading to the identification of more than 1000 proteins from each microdissected piece. The strength of this cheap technique lies in the facile excision of millimeter-sized portions from the tissue allowing for the identification of proteins from cells of a specific phenotype obtained from the MALDI MS imaging-based molecular classification using hierarchical clustering. This approach can be extended to whole tissue sections in order to generate images of the section based on label-free quantification obtained from identification data. As a proof of concept, we have studied a tissue mounted on a parafilm M-covered glass slide, cut it into regular pieces, and submitted each piece to identification and quantification according to the developed parafilm-assisted microdissection (PAM) method. Images were then reconstructed by relative quantification of identified proteins based on spectral counting of the peptides analyzed by nanoLC-MS and MS/MS. This strategy of quantification-based MSI offers new possibilities for mapping a large number of high and low abundance proteins.

  15. Setting reference targets

    SciTech Connect

    Ruland, R.E.

    1997-04-01

    Reference Targets are used to represent virtual quantities like the magnetic axis of a magnet or the definition of a coordinate system. To explain the function of reference targets in the sequence of the alignment process, this paper will first briefly discuss the geometry of the trajectory design space and of the surveying space, then continue with an overview of a typical alignment process. This is followed by a discussion on magnet fiducialization. While the magnetic measurement methods to determine the magnetic centerline are only listed (they will be discussed in detail in a subsequent talk), emphasis is given to the optical/mechanical methods and to the task of transferring the centerline position to reference targets.

  16. Feasibility study for the quantification of total protein content by multiple prompt gamma-ray analysis.

    PubMed

    Toh, Y; Murakami, Y; Furutaka, K; Kimura, A; Koizumi, M; Hara, K; Kin, T; Nakamura, S; Harada, H

    2012-06-01

    Protein is an important nutrient in foods. The classical nitrogen analysis method is the Kjeldahl technique, which is time-consuming and inconvenient. As a convenient method to quantify protein content in biological samples, the feasibility of application of multiple prompt gamma-ray analysis (MPGA) to the quantification was studied. Results for protein content are reported for several reference materials and prove the method to be reliable.

  17. Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

    PubMed Central

    Rajendran, Simon; Salwa, Slawomir; Gao, Xuefeng; Tabirca, Sabin; O'Hanlon, Deirdre; O'Sullivan, Gerald C.; Tangney, Mark

    2010-01-01

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial. PMID:21326169

  18. Multifunctional reference electrode

    DOEpatents

    Redey, L.; Vissers, D.R.

    1981-12-30

    A multifunctional, low mass reference electrode of a nickel tube, thermocouple means inside the nickel tube electrically insulated therefrom for measuring the temperature thereof, a housing surrounding the nickel tube, an electrolyte having a fixed sulfide ion activity between the housing and the outer surface of the nickel tube forming the nickel/nickel sulfide/sulfide half-cell are described. An ion diffusion barrier is associated with the housing in contact with the electrolyte. Also disclosed is a cell using the reference electrode to measure characteristics of a working electrode.

  19. Multifunctional reference electrode

    DOEpatents

    Redey, Laszlo; Vissers, Donald R.

    1983-01-01

    A multifunctional, low mass reference electrode of a nickel tube, thermocou