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Sample records for reference tissue quantification

  1. Reference tissue quantification of DCE-MRI data without a contrast agent calibration

    NASA Astrophysics Data System (ADS)

    Walker-Samuel, Simon; Leach, Martin O.; Collins, David J.

    2007-02-01

    The quantification of dynamic contrast-enhanced (DCE) MRI data conventionally requires a conversion from signal intensity to contrast agent concentration by measuring a change in the tissue longitudinal relaxation rate, R1. In this paper, it is shown that the use of a spoiled gradient-echo acquisition sequence (optimized so that signal intensity scales linearly with contrast agent concentration) in conjunction with a reference tissue-derived vascular input function (VIF), avoids the need for the conversion to Gd-DTPA concentration. This study evaluates how to optimize such sequences and which dynamic time-series parameters are most suitable for this type of analysis. It is shown that signal difference and relative enhancement provide useful alternatives when full contrast agent quantification cannot be achieved, but that pharmacokinetic parameters derived from both contain sources of error (such as those caused by differences between reference tissue and region of interest proton density and native T1 values). It is shown in a rectal cancer study that these sources of uncertainty are smaller when using signal difference, compared with relative enhancement (15 ± 4% compared with 33 ± 4%). Both of these uncertainties are of the order of those associated with the conversion to Gd-DTPA concentration, according to literature estimates.

  2. Validation of RPS13 as a reference gene for absolute quantification of SIV RNA in tissue of rhesus macaques.

    PubMed

    Robichaux, Spencer; Lacour, Nedra; Bagby, Gregory J; Amedee, Angela M

    2016-10-01

    Persistent HIV reservoirs and the absolute quantification of viral RNA copies in tissues have become a prominent focus of multiple areas ofHIV/SIV research. Absolute quantification of viral RNA via reverse transcription, quantitative PCR (RT-qPCR) necessitates the use of an appropriate RNA reference gene whose expression is unaffected by both experimental and confounding conditions. In this study, we demonstrate the utility of ribosomal protein S13 mRNA (RPS13) as a stable, medium abundance reference gene for RT-qPCR normalization of HIV/SIV RNA copy number. We developed a RPS13 RNA standard assay utilizing an in vitro RNA transcript for normalization of absolute SIV RNA quantities in tissues reservoirs. The RT-qPCR assay showed a high degree of repeatability and reproducibility across RNA levels appropriate for absolute SIV quantification. In assessing the utility of RPS13 as a reference gene, limited variation in the absolute, inter-tissue quantities of RPS13 mRNA was observed within multiple tissue samples obtained from rhesus macaques (average CV=2.86%). We demonstrate rhesus macaque RPS13 mRNA expression is not affected by alcohol administration, SIV infection, or antiviral therapy (PMPA/FTC). Additionally, assay functionality was validated for normalization of SIV copy number using cellular RNA prepared from samples of variable RNA integrity. RPS13 is a suitable reference gene for normalization of absolute SIV RNA quantities in tissues and is most appropriate for intra-tissue or similar tissue type comparisons of SIV copy number. PMID:27510462

  3. Validation of RPS13 as a reference gene for absolute quantification of SIV RNA in tissue of rhesus macaques.

    PubMed

    Robichaux, Spencer; Lacour, Nedra; Bagby, Gregory J; Amedee, Angela M

    2016-10-01

    Persistent HIV reservoirs and the absolute quantification of viral RNA copies in tissues have become a prominent focus of multiple areas ofHIV/SIV research. Absolute quantification of viral RNA via reverse transcription, quantitative PCR (RT-qPCR) necessitates the use of an appropriate RNA reference gene whose expression is unaffected by both experimental and confounding conditions. In this study, we demonstrate the utility of ribosomal protein S13 mRNA (RPS13) as a stable, medium abundance reference gene for RT-qPCR normalization of HIV/SIV RNA copy number. We developed a RPS13 RNA standard assay utilizing an in vitro RNA transcript for normalization of absolute SIV RNA quantities in tissues reservoirs. The RT-qPCR assay showed a high degree of repeatability and reproducibility across RNA levels appropriate for absolute SIV quantification. In assessing the utility of RPS13 as a reference gene, limited variation in the absolute, inter-tissue quantities of RPS13 mRNA was observed within multiple tissue samples obtained from rhesus macaques (average CV=2.86%). We demonstrate rhesus macaque RPS13 mRNA expression is not affected by alcohol administration, SIV infection, or antiviral therapy (PMPA/FTC). Additionally, assay functionality was validated for normalization of SIV copy number using cellular RNA prepared from samples of variable RNA integrity. RPS13 is a suitable reference gene for normalization of absolute SIV RNA quantities in tissues and is most appropriate for intra-tissue or similar tissue type comparisons of SIV copy number.

  4. [F-18]-Fluoromisonidazole Quantification of Hypoxia in Human Cancer Patients using Image-derived Blood Surrogate Tissue Reference Regions

    PubMed Central

    Muzi, Mark; Peterson, Lanell M.; O’Sullivan, Janet N.; Fink, James R.; Rajendran, Joseph G.; McLaughlin, Lena J.; Muzi, John P.; Mankoff, David A.; Krohn, Kenneth A.

    2015-01-01

    18F-FMISO is the most widely used PET agent for imaging hypoxia, a condition associated with resistance to tumor therapy. 18F-FMISO equilibrates in normoxic tissues, but is retained under hypoxic conditions because of reduction and binding to macromolecules. A simple tissue-to-blood ratio (TB) is suitable for quantifying hypoxia. A threshold of TB ≥ 1.2 is useful in discriminating the hypoxic volume (HV) of tissue; TBmax is the maximum intensity of the hypoxic region and does not invoke a threshold. Because elimination of blood sampling would simplify clinical use, we tested the validity of using imaging regions as a surrogate for blood sampling. Methods Patients underwent 20 min 18F-FMISO scans during the 90–140 min interval post-injection with venous blood sampling. 223 18F-FMISO patient studies had detectable surrogate blood regions in the field-of-view. Quantitative parameters of hypoxia (TBmax, HV) derived from blood samples were compared to values using surrogate blood regions derived from heart, aorta and/or cerebellum. In a subset of brain cancer patients, parameters from blood samples and from cerebellum were compared for their ability to independently predict outcome. Results Vascular regions of heart showed the highest correlation to measured blood activity (R2 = 0.84). For brain studies, cerebellar activity was similarly correlated to blood samples. In brain cancer patients, Kaplan-Meier analysis showed that image-derived reference regions had nearly identical predictive power as parameters derived from blood, thus obviating the need for venous sampling in these patients. Conclusions Simple static analysis of 18F-FMISO PET captures both the intensity (TBmax) and spatial extent (HV) of tumor hypoxia. An image-derived region to assess blood activity can be used as a surrogate for blood sampling in quantification of hypoxia. PMID:26112020

  5. Selection of weighting factors for quantification of PET radioligand binding using simplified reference tissue models with noisy input functions.

    PubMed

    Normandin, M D; Koeppe, R A; Morris, E D

    2012-02-01

    Input function noise contributes to model-predicted values and should be accounted for during parameter estimation. This problem has been examined in the context of PET data analysis using a noisy image-derived arterial input function. Huesman and Mazoyer (1987 Phys. Med. Biol 32 1569-79) incorporated the effect of error in the measured input function into the objective function and observed a subsequent improvement in the accuracy of parameters estimated from a kinetic model of cardiac blood flow. Such a treatment has not been applied to the reference region models commonly used to analyze dynamic positron emission tomography data with receptor-ligand tracers. Here, we propose a strategy for selection of weighting factors that accounts for noise in the reference region input function and test the method on two common formulations of the simplified reference tissue model (SRTM). We present a simulation study which demonstrates that the proposed weighting approach improves the accuracy of estimated binding potential at high noise levels and when the reference tissue and target regions of interest are of comparable size. In the second simulation experiment, we show that using a small, homogeneous reference tissue with our weighting technique may have advantages over input functions derived from a larger (and thus less noisy), heterogeneous region with conventional weighting. A comparative analysis of clinical [(11)C]flumazenil data found a small but significant increase in estimated binding potential when using the proposed weighting method, consistent with the finding of reduced negative bias in our simulation study. The weighting strategy described here accounts for noise in the reference region input function and may improve the performance of the SRTM in applications where data are noisy and the reference region is relatively small. This technique may offer similar benefits to other models using reference region inputs, particularly those derived from the SRTM

  6. A modified simplified reference tissue model for the quantification of dopamine D2/3 receptors with [18F]fallypride images.

    PubMed

    Tsartsalis, Stergios; Moulin-Sallanon, Marcelle; Dumas, Noé; Tournier, Benjamin B; Ginovart, Nathalie; Millet, Philippe

    2014-01-01

    Defluorination of [18F]fallypride and accumulation of 18F in skull and glands leads to the contamination of brain structures with spillover activity due to partial volume effects, leading to considerable errors in binding potential estimations. Here we propose a modification of the simplified reference tissue model (SRTM) to take into account the contribution of skull activity to the radioactivity kinetic pattern in cerebellum and target regions. It consists of the introduction of an additional parameter for each volume of interest (sT) and one for the cerebellum (sR), corresponding to the fraction of skull activity contaminating these structures. Using five rat positron emission tomography experiments, we applied the modified SRTM (SRTMc), which resulted in excellent fits. As a relative means of comparison of results, we applied factor analysis (FA) to decompose dynamic data into images corresponding to brain and skull activity. With the skull factor images, we estimated the "true" sT and sR values, ultimately permitting us to fix the sR value. Parameters obtained with the SRTMc were closely correlated with values obtained from FA-corrected data. In conclusion, we propose an efficient method for reliable quantification of dopamine D2/3 receptors with single-injection [18F]fallypride scans that is potentially applicable to human studies where 18F skull accumulation compromises binding parameter estimation. PMID:25248453

  7. In Vivo Evaluation of Blood Based and Reference Tissue Based PET Quantifications of [11C]DASB in the Canine Brain

    PubMed Central

    Polis, Ingeborgh; Neyt, Sara; Kersemans, Ken; Dobbeleir, Andre; Saunders, Jimmy; Goethals, Ingeborg; Peremans, Kathelijne; De Vos, Filip

    2016-01-01

    This first-in-dog study evaluates the use of the PET-radioligand [11C]DASB to image the density and availability of the serotonin transporter (SERT) in the canine brain. Imaging the serotonergic system could improve diagnosis and therapy of multiple canine behavioural disorders. Furthermore, as many similarities are reported between several human neuropsychiatric conditions and naturally occurring canine behavioural disorders, making this tracer available for use in dogs also provide researchers an interesting non-primate animal model to investigate human disorders. Five adult beagles underwent a 90 minutes dynamic PET scan and arterial whole blood was sampled throughout the scan. For each ROI, the distribution volume (VT), obtained via the one- and two- tissue compartment model (1-TC, 2-TC) and the Logan Plot, was calculated and the goodness-of-fit was evaluated by the Akaike Information Criterion (AIC). For the preferred compartmental model BPND values were estimated and compared with those derived by four reference tissue models: 4-parameter RTM, SRTM2, MRTM2 and the Logan reference tissue model. The 2-TC model indicated in 61% of the ROIs a better fit compared to the 1-TC model. The Logan plot produced almost identical VT values and can be used as an alternative. Compared with the 2-TC model, all investigated reference tissue models showed high correlations but small underestimations of the BPND-parameter. The highest correlation was achieved with the Logan reference tissue model (Y = 0.9266 x + 0.0257; R2 = 0.9722). Therefore, this model can be put forward as a non-invasive standard model for future PET-experiments with [11C]DASB in dogs. PMID:26859850

  8. In Vivo Evaluation of Blood Based and Reference Tissue Based PET Quantifications of [11C]DASB in the Canine Brain.

    PubMed

    Van Laeken, Nick; Taylor, Olivia; Polis, Ingeborgh; Neyt, Sara; Kersemans, Ken; Dobbeleir, Andre; Saunders, Jimmy; Goethals, Ingeborg; Peremans, Kathelijne; De Vos, Filip

    2016-01-01

    This first-in-dog study evaluates the use of the PET-radioligand [11C]DASB to image the density and availability of the serotonin transporter (SERT) in the canine brain. Imaging the serotonergic system could improve diagnosis and therapy of multiple canine behavioural disorders. Furthermore, as many similarities are reported between several human neuropsychiatric conditions and naturally occurring canine behavioural disorders, making this tracer available for use in dogs also provide researchers an interesting non-primate animal model to investigate human disorders. Five adult beagles underwent a 90 minutes dynamic PET scan and arterial whole blood was sampled throughout the scan. For each ROI, the distribution volume (VT), obtained via the one- and two- tissue compartment model (1-TC, 2-TC) and the Logan Plot, was calculated and the goodness-of-fit was evaluated by the Akaike Information Criterion (AIC). For the preferred compartmental model BPND values were estimated and compared with those derived by four reference tissue models: 4-parameter RTM, SRTM2, MRTM2 and the Logan reference tissue model. The 2-TC model indicated in 61% of the ROIs a better fit compared to the 1-TC model. The Logan plot produced almost identical VT values and can be used as an alternative. Compared with the 2-TC model, all investigated reference tissue models showed high correlations but small underestimations of the BPND-parameter. The highest correlation was achieved with the Logan reference tissue model (Y = 0.9266 x + 0.0257; R2 = 0.9722). Therefore, this model can be put forward as a non-invasive standard model for future PET-experiments with [11C]DASB in dogs. PMID:26859850

  9. Quantification of adipose tissue insulin sensitivity.

    PubMed

    Søndergaard, Esben; Jensen, Michael D

    2016-06-01

    In metabolically healthy humans, adipose tissue is exquisitely sensitive to insulin. Similar to muscle and liver, adipose tissue lipolysis is insulin resistant in adults with central obesity and type 2 diabetes. Perhaps uniquely, however, insulin resistance in adipose tissue may directly contribute to development of insulin resistance in muscle and liver because of the increased delivery of free fatty acids to those tissues. It has been hypothesized that insulin adipose tissue resistance may precede other metabolic defects in obesity and type 2 diabetes. Therefore, precise and reproducible quantification of adipose tissue insulin sensitivity, in vivo, in humans, is an important measure. Unfortunately, no consensus exists on how to determine adipose tissue insulin sensitivity. We review the methods available to quantitate adipose tissue insulin sensitivity and will discuss their strengths and weaknesses.

  10. 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.

  11. 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.

  12. Quantification of brodifacoum in plasma and liver tissue by HPLC.

    PubMed

    O'Bryan, S M; Constable, D J

    1991-01-01

    A simple high-performance liquid chromatographic method has been developed for detection and quantification of brodifacoum in plasma and liver tissue. After adding difenacoum as the internal standard, brodifacoum and difenacoum are extracted from 2 mL of plasma with two sequential 10-mL volumes of acetonitrile-ethyl ether (9:1) and from 2 g of liver tissue by grinding the tissue with 10 mL acetonitrile. The extracts are evaporated to dryness under nitrogen, 2 mL of acetonitrile is added to reconstitute the residues, and the resulting solution is analyzed using reversed-phase chromatography and fluorescence detection. The limits of detection for plasma and tissue are 2 micrograms/L and 5 ng/g, respectively. Using internal standardization, the mean intra-assay recovery from plasma is 92% and the mean inter-assay recoveries is 109%. The mean intra-assay and inter-assay recoveries from tissue are 96%. No interferences were observed with any of the following related compounds: brodifacoum, bromadiolone, coumarin, difenacoum, diphacinone, warfarin, and vitamin K1. PMID:1943058

  13. 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

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

    PubMed

    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.

  15. Quantification of phospholipids in excised tissues by NMR

    SciTech Connect

    Barany, M.; Venkatasubramanian, P.N.

    1986-05-01

    A fraction of the total phospholipids is visible in natural abundance /sup 13/C NMR spectra of diseased human muscle biopsies which have been extracted with isopentane to remove neutral fats. The authors have quantified the visible phospholipids by inserting into the muscle biopsies a dioxane capillary which was calibrated against phospholipid vesicles (with known phosphate concentration) prepared from rat muscle and liver, and against pure palmitic and linolenic acid. The phospholipid content of the human muscles was calculated from the integrated peak area of the dioxane capillary, from the area of the 30.5 and 128.5 ppm peaks in the /sup 13/C spectrum of the muscle, and from the dry weight of the muscle, determined on the same sample which was used for /sup 13/C spectroscopy. The same experiments were carried out with rat muscle, brain, liver, and kidney. Furthermore, after the /sup 13/C spectrum of the tissue was recorded excess halothane was added into the NMR tube, the sample incubated, then the /sup 13/C spectrum re-recorded for quantification of the total phospholipids. Thus, their procedure quantitates both the NMR visible and the total phospholipids of the tissue. The total phospholipid content determined by NMR was in good agreement with that determined by chemical analysis.

  16. En route to traceable reference standards for surface group quantifications by XPS, NMR and fluorescence spectroscopy.

    PubMed

    Hennig, Andreas; Dietrich, Paul M; Hemmann, Felix; Thiele, Thomas; Borcherding, Heike; Hoffmann, Angelika; Schedler, Uwe; Jäger, Christian; Resch-Genger, Ute; Unger, Wolfgang E S

    2015-03-21

    The fluorine content of polymer particles labelled with 2,2,2-trifluoroethylamine was reliably quantified with overlapping sensitivity ranges by XPS and solid-state NMR. This provides a first step towards reference materials for the metrological traceability of surface group quantifications. The extension of this concept to fluorescence spectroscopy is illustrated.

  17. Quantification of adipose tissue in a rodent model of obesity

    NASA Astrophysics Data System (ADS)

    Johnson, David H.; Flask, Chris; Wan, Dinah; Ernsberger, Paul; Wilson, David L.

    2006-03-01

    Obesity is a global epidemic and a comorbidity for many diseases. We are using MRI to characterize obesity in rodents, especially with regard to visceral fat. Rats were scanned on a 1.5T clinical scanner, and a T1W, water-spoiled image (fat only) was divided by a matched T1W image (fat + water) to yield a ratio image related to the lipid content in each voxel. The ratio eliminated coil sensitivity inhomogeneity and gave flat values across a fat pad, except for outlier voxels (> 1.0) due to motion. Following sacrifice, fat pad volumes were dissected and measured by displacement in canola oil. In our study of 6 lean (SHR), 6 dietary obese (SHR-DO), and 9 genetically obese rats (SHROB), significant differences in visceral fat volume was observed with an average of 29+/-16 ml increase due to diet and 84+/-44 ml increase due to genetics relative to lean control with a volume of 11+/-4 ml. Subcutaneous fat increased 14+/-8 ml due to diet and 198+/-105 ml due to genetics relative to the lean control with 7+/-3 ml. Visceral fat strongly correlated between MRI and dissection (R2 = 0.94), but MRI detected over five times the subcutaneous fat found with error-prone dissection. Using a semi-automated images segmentation method on the ratio images, intra-subject variation was very low. Fat pad composition as estimated from ratio images consistently differentiated the strains with SHROB having a greater lipid concentration in adipose tissues. Future work will include in vivo studies of diet versus genetics, identification of new phenotypes, and corrective measures for obesity; technical efforts will focus on correction for motion and automation in quantification.

  18. Validation of Reference Genes in Solenopsis invicta in Different Developmental Stages, Castes and Tissues

    PubMed Central

    Cheng, Daifeng; Zhang, Zhiling; He, Xiaofang; Liang, Guangwen

    2013-01-01

    To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta. PMID:23469057

  19. Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

    PubMed Central

    Wisnieski, Fernanda; Calcagno, Danielle Queiroz; Leal, Mariana Ferreira; dos Santos, Leonardo Caires; Gigek, Carolina de Oliveira; Chen, Elizabeth Suchi; Pontes, Thaís Brilhante; Assumpção, Paulo Pimentel; de Assumpção, Mônica Barauna; Demachki, Sâmia; Burbano, Rommel Rodríguez; Smith, Marília de Arruda Cardoso

    2013-01-01

    AIM: To evaluate the suitability of reference genes in gastric tissue samples and cell lines. METHODS: The suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent non-neoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The ranking of the best single and combination of reference genes was determined by NormFinder, geNorm™, BestKeeper, and DataAssist™. In addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization. RESULTS: ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. The level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44). CONCLUSION: GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested. PMID:24222956

  20. Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.

    PubMed

    Yu, Hannah; Hahn, Yoonsoo; Yang, Inchul

    2015-01-01

    Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements. PMID:26368560

  1. Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.

    PubMed

    Mehta, Nickita; Porterfield, Mindy; Struwe, Weston B; Heiss, Christian; Azadi, Parastoo; Rudd, Pauline M; Tiemeyer, Michael; Aoki, Kazuhiro

    2016-09-01

    Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards. PMID:27432553

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

    PubMed

    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

  3. 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

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

    PubMed

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

    2016-09-15

    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.

  5. 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

  6. 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

  7. Single point quantification of antibody by ELISA without need of a reference curve.

    PubMed

    Dopatka, H D; Giesendorf, B

    1992-01-01

    A new method for the quantification of antibodies in the enzyme-linked immunosorbent assay is described. This procedure replaces titer determinations based on end-point dilution of the antibody under investigation. Here, the sample is tested in a single dilution and the optical density (OD) obtained is used in the equation log10 titer = alpha.OD beta. The titer can then be calculated by inserting into the formula the values for the constants alpha and beta, which are specified by the manufacturer for each separate batch of kit reagents. This so-called alpha-method saves time and reagents while providing results which are equal to the titration method in accuracy and superior in precision. The alpha-method is also a simpler and reliable alternative to the use of standard or reference curves for the quantification of the antibodies in I.U./ml.

  8. Selection of reference genes in canine uterine tissues.

    PubMed

    Du, M; Wang, X; Yue, Y W; Zhou, P Y; Yao, W; Li, X; Ding, X B; Liu, X F; Guo, H; Ma, W Z

    2016-01-01

    Real-time quantitative polymerase chain reaction (RT-qPCR) is usually employed in gene expression studies in veterinary research, including in studies on canine pyometra. Canine pyometra is a common clinical disease in bitches. When using RT-qPCR, internal standards, such as reference genes, are necessary to investigate relative gene expression by quantitative measurements of mRNA levels. The aim of this study was to evaluate the stability of reference genes and select reference genes suitable for canine pyometra studies. We collected 24 bitch uterine tissue samples, including five healthy and 19 pyometra infected samples. These were used to screen the best reference genes of seven candidate genes (18SrRNA, ACTB, B2M, GAPDH, HPRT, RPL13A, and YWHAZ). The method of KH Sadek and the GeNorm, Normfinder, BestKeeper, and RefFinder software were used to evaluate the stability of gene expression in both pyometra and healthy uterine samples. The results showed that the expression stability of the candidate gene in pyometra and healthy tissues differed. We showed that YWHAZ was the best reference gene, which could be used as an accurate internal control gene in canine pyometra studies. To further validate this recommendation, the expression profile of a target gene insulin-like growth factor 1 receptor gene (IGF1R) was investigated. We found that the expression of IGF1R was significantly altered when different reference genes were used. All reference genes identified in the present study will enable more accurate normalization of gene expression data in both pyometra infected and healthy uterine tissues. PMID:27323194

  9. Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods.

    PubMed

    Cao, Yiping; Sivaganesan, Mano; Kinzelman, Julie; Blackwood, A Denene; Noble, Rachel T; Haugland, Richard A; Griffith, John F; Weisberg, Stephen B

    2013-01-01

    Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an option for recreational water quality testing in the United States (USEPA, 2011. EPA-OW-2011-0466, FRL-9609-3, Notice of Availability of Draft Recreational Water Quality Criteria and Request for Scientific Views). However, transition of qPCR from a research tool to routine water quality testing requires information on how various method variations affect target enumeration. Here we compared qPCR performance and enumeration of enterococci in spiked and environmental water samples using three qPCR platforms (Applied Biosystem StepOnePlus™, the BioRad iQ™5 and the Cepheid SmartCycler(®) II), two reference materials (lyophilized cells and frozen cells on filters) and two comparative CT quantification models (ΔCT and ΔΔCT). Reference materials exerted the biggest influence, consistently affecting results by approximately 0.5 log(10) unit. Platform had the smallest effect, generally exerting <0.1 log(10) unit difference in final results. Quantification model led to small differences (0.04-0.2 log(10) unit) in this study with relatively uninhibited samples, but has the potential to cause as much as 8-fold (0.9 log(10) unit) difference in potentially inhibitory samples. Our findings indicate the need for a certified and centralized source of reference materials and additional studies to assess applicability of the quantification models in analyses of PCR inhibitory samples.

  10. 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.

  11. Considerations for quantification of lipids in nerve tissue using MALDI mass spectrometric imaging

    PubMed Central

    Landgraf, Rachelle R.; Garrett, Timothy J.; Prieto Conaway, Maria C.; Calcutt, Nigel A.; Stacpoole, Peter W.; Yost, Richard A.

    2013-01-01

    MALDI mass spectrometric imaging is a technique that provides the ability to identify and characterize endogenous and exogenous compounds spatially within tissue with relatively little sample preparation. While it is a proven methodology for qualitative analysis, little has been reported for its utility in quantitative measurements. In the current work, inherent challenges in MALDI quantification are addressed. Signal response is monitored over successive analyses of a single tissue section to minimize error due to variability in the laser, matrix application, and sample inhomogeneity. Methods for the application of an internal standard to tissue sections are evaluated and used to quantify endogenous lipids in nerve tissue. A precision of 5% or less standard error was achieved, illustrating that MALDI imaging offers a reliable means of in situ quantification for microgram-sized samples and requires minimal sample preparation. PMID:21953974

  12. 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.

  13. 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.

  14. 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

  15. Nondestructive quantification of permeability of hyperosmotic agent in normal and tumor tissues

    NASA Astrophysics Data System (ADS)

    Xiong, Honglian; Guo, Zhouyi; Zeng, Changchun; Wang, Like; He, Yonghong; Liu, Songhao

    2008-12-01

    Noninvasive tumor imaging could lead to the early detection and timely treatment of cancer. Previous investigations have suggested that optical coherence tomography (OCT) is an ideal diagnostic tool distinguishing normal tissues from tumor tissues based on structural imaging because of the high resolution. In the study, the capability of OCT for functional imaging of normal and tumor tissues based on time and depth resolved quantification of the permeability of biomolecules through these tissues is investigated. An OCT system at 830 nm central wavelength was used in this study. Diffusion of 20% aqueous solution of glucose was monitored and quantified in normal stomach tissues and tumor tissues using OCT. The orthotopic graft model of gastric cancer in nude mice was used. Permeability coefficients were calculated as a function of time and tissue depth. The permeability coefficient was (9.44+/-0.42) ×10-6 cm/s in normal stomach tissues and (5.32+/-0.17)×10-5 cm/s in tumor tissues. The tumor tissues had a higher permeability coefficient compared to normal tissues. From the experimental results, it is found that the accurate and sensitive assessment of the permeability coefficients of normal and tumor tissues offer an effective OCT image method for clinical diagnosis and detection of tumor tissues.

  16. 18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry.

    PubMed

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

    2011-12-01

    Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

  17. Tissue viability imaging for quantification of skin erythema and blanching

    NASA Astrophysics Data System (ADS)

    Nilsson, Gert E.; Leahy, Martin J.

    2010-02-01

    Naked eye observation has up to recently been the main method of determining skin erythema (vasodilatation) and blanching (vasoconstriction) in skin testing. Since naked eye observation is a highly subjective and investigatordependent method, it is difficult to attain reproducibility and to compare results reported by different researchers performing their studies at different laboratories. Consequently there is a need for more objective, quantitative and versatile methods in the assessment of alterations in skin erythema and blanching caused by internal and external factors such as the intake of vasoactive drugs, application of agents on the skin surface and by constituents in the environment. Since skin microcirculation is sensitive to applied pressure and heat, such methods should preferably be noninvasive and designed for remote use without touching the skin. As skin microcirculation further possesses substantial spatial variability, imaging techniques are to be preferred before single point measurements. An emerging technology based on polarization digital camera spectroscopy - Tissue Viability Imaging (TiVi) - fulfills these requirements. The principles of TiVi (1) and some of its early applications (2-5) are addressed in this paper.

  18. Quantification of titanium from TiO2 particles in biological tissue.

    PubMed

    Faucher, Stéphane; Lespes, Gaëtane

    2015-10-01

    This study presents the development of a strategy for the quantification of titanium from titanium dioxide polydisperse particles (TiO2) in dry biological tissue. Calf liver was chosen as laboratory testing material. The challenge was to (i) obtain a complete mineralization of the solid material (biological tissue and TiO2) and (ii) ensure the accuracy of the determined concentrations with a sufficient sensitivity. Mineralization was performed using a mixture of concentrated nitric and hydrofluoric acids. Atomic mass spectrometry associated with light-scattering technique was used to control the physical state (dissolved and particle forms) of titanium and reliably estimate the total titanium concentration in calf liver. The monitoring of (46)Ti and (49)Ti, operating in helium collision/reaction cell mode, and using external calibration with internal standard addition, allowed the quantification of Ti while removing isobaric interferences. The limit of detection and quantification were 0.7 and 2.3μg (Ti)g(-1) (tissue) respectively. The mean analytical recovery over the whole procedure was (103±6)% in a range of concentrations from LOD to 200μg(Ti)g(-1) (tissue). PMID:26302910

  19. Quantification of titanium from TiO2 particles in biological tissue.

    PubMed

    Faucher, Stéphane; Lespes, Gaëtane

    2015-10-01

    This study presents the development of a strategy for the quantification of titanium from titanium dioxide polydisperse particles (TiO2) in dry biological tissue. Calf liver was chosen as laboratory testing material. The challenge was to (i) obtain a complete mineralization of the solid material (biological tissue and TiO2) and (ii) ensure the accuracy of the determined concentrations with a sufficient sensitivity. Mineralization was performed using a mixture of concentrated nitric and hydrofluoric acids. Atomic mass spectrometry associated with light-scattering technique was used to control the physical state (dissolved and particle forms) of titanium and reliably estimate the total titanium concentration in calf liver. The monitoring of (46)Ti and (49)Ti, operating in helium collision/reaction cell mode, and using external calibration with internal standard addition, allowed the quantification of Ti while removing isobaric interferences. The limit of detection and quantification were 0.7 and 2.3μg (Ti)g(-1) (tissue) respectively. The mean analytical recovery over the whole procedure was (103±6)% in a range of concentrations from LOD to 200μg(Ti)g(-1) (tissue).

  20. The simplified reference tissue model: model assumption violations and their impact on binding potential.

    PubMed

    Salinas, Cristian A; Searle, Graham E; Gunn, Roger N

    2015-02-01

    Reference tissue models have gained significant traction over the last two decades as the methods of choice for the quantification of brain positron emission tomography data because they balance quantitative accuracy with less invasive procedures. The principal advantage is the elimination of the need to perform arterial cannulation of the subject to measure blood and metabolite concentrations for input function generation. In particular, the simplified reference tissue model (SRTM) has been widely adopted as it uses a simplified model configuration with only three parameters that typically produces good fits to the kinetic data and a stable parameter estimation process. However, the model's simplicity and its ability to generate good fits to the data, even when the model assumptions are not met, can lead to misplaced confidence in binding potential (BPND) estimates. Computer simulation were used to study the bias introduced in BPND estimates as a consequence of violating each of the four core SRTM model assumptions. Violation of each model assumption led to bias in BPND (both over and underestimation). Careful assessment of the bias in SRTM BPND should be performed for new tracers and applications so that an appropriate decision about its applicability can be made. PMID:25425078

  1. Anisotropic Properties of Breast Tissue Measured by Acoustic Radiation Force Impulse Quantification.

    PubMed

    Zhou, JianQiao; Yang, ZhiFang; Zhan, WeiWei; Dong, YiJie; Zhou, Chun

    2016-10-01

    The goal of our study was to investigate the anisotropy of normal breast glandular and fatty tissue with acoustic radiation force impulse (ARFI) quantification. A total of 137 breasts in 137 women were enrolled. These breasts were divided into the duct-apparent group and the duct-inapparent group, divided into the ligament-apparent group and the ligament-inapparent group. Shear wave velocity (SWV) in the radial (SWV(r)) and anti-radial (SWV(a-r)) directions was measured. The elastic anisotropy of glandular tissue and fatty tissue was evaluated as the ratio between SWV(r) and SWV(a-r). The SWV ratio was 1.30 ± 0.45 for glandular tissue and 1.27 ± 0.53 for fatty tissue in the total group. In glandular tissue, the SWV ratio of the duct-apparent group was higher than that of the duct-inapparent group (p = 0.011). In both glandular and fatty tissue, the SWV ratio was higher in the ligament-apparent group than in the ligament-inapparent group (p < 0.05 for both). SWV(r) was higher than SWV(a-r) in both glandular tissue and fatty tissue in all groups (p < 0.05 for all) except in breast fatty tissue in the ligament-inapparent group (p = 0.913). It is concluded that both breast glandular tissue and fatty tissue exhibited anisotropy of elastic behavior. To improve the diagnostic power of elastography in breast lesions, the elastic anisotropy of glandular tissue and fatty tissue should be taken into account in calculating strain ratio or elasticity ratio.

  2. Anisotropic Properties of Breast Tissue Measured by Acoustic Radiation Force Impulse Quantification.

    PubMed

    Zhou, JianQiao; Yang, ZhiFang; Zhan, WeiWei; Dong, YiJie; Zhou, Chun

    2016-10-01

    The goal of our study was to investigate the anisotropy of normal breast glandular and fatty tissue with acoustic radiation force impulse (ARFI) quantification. A total of 137 breasts in 137 women were enrolled. These breasts were divided into the duct-apparent group and the duct-inapparent group, divided into the ligament-apparent group and the ligament-inapparent group. Shear wave velocity (SWV) in the radial (SWV(r)) and anti-radial (SWV(a-r)) directions was measured. The elastic anisotropy of glandular tissue and fatty tissue was evaluated as the ratio between SWV(r) and SWV(a-r). The SWV ratio was 1.30 ± 0.45 for glandular tissue and 1.27 ± 0.53 for fatty tissue in the total group. In glandular tissue, the SWV ratio of the duct-apparent group was higher than that of the duct-inapparent group (p = 0.011). In both glandular and fatty tissue, the SWV ratio was higher in the ligament-apparent group than in the ligament-inapparent group (p < 0.05 for both). SWV(r) was higher than SWV(a-r) in both glandular tissue and fatty tissue in all groups (p < 0.05 for all) except in breast fatty tissue in the ligament-inapparent group (p = 0.913). It is concluded that both breast glandular tissue and fatty tissue exhibited anisotropy of elastic behavior. To improve the diagnostic power of elastography in breast lesions, the elastic anisotropy of glandular tissue and fatty tissue should be taken into account in calculating strain ratio or elasticity ratio. PMID:27471118

  3. Ultra-trace quantification method for chlordecone in human fluids and tissues.

    PubMed

    Bichon, Emmanuelle; Guiffard, Ingrid; Vénisseau, Anaïs; Marchand, Philippe; Antignac, Jean-Philippe; Le Bizec, Bruno

    2015-08-21

    Chlordecone is an organochlorine pesticide (OCP) considered as a Persistent Organic Pollutant (POP) as it persists in the environment, bio-accumulates through the food web, causes adverse effects to human health and the environment and transports across international boundaries far from its sources. The atypical physico-chemical properties of chlordecone make its inclusion in classical analytical approaches non applicable. The aim of our work was to include chlordecone in a multi organochlorine residue method preventing any degradation during the analytical process and thus allowing quantification at ppt (ngkg(-1) or ngL(-1)) levels for a wide range of OCPs in breast milk, human serum and adipose tissue. After GC-HRMS vs. MS/MS and EI vs. APCI comparisons, the major improvement in terms of sensitivity was found in decreasing the length and film thickness of the gas chromatography column. Thanks to a linear correlation between relative response and quantity of chlordecone injected, LC-(ESI-)-MS/MS was finally preferred. An acetonitrile based gradient optimized on a C30 coreshell HPLC column has led to reaching limits of quantification as low as 8ngL(-1), 25pgmL(-1) and 0.2ngg(-1) fat for breast milk, serum and adipose tissue, respectively, allowing multiresidue OCP quantification at concentration levels compatible with biomonitoring purposes and pre-requisites. PMID:26184709

  4. Tissue elasticity quantification by acoustic radiation force impulse for the assessment of renal allograft function.

    PubMed

    He, Wan-Yuan; Jin, Yun-Jie; Wang, Wen-Ping; Li, Chao-Lun; Ji, Zheng-Biao; Yang, Cheng

    2014-02-01

    Acoustic radiation force impulse (ARFI) quantification, a novel ultrasound-based elastography method, has been used to measure liver fibrosis. However, few studies have been performed on the use of ARFI quantification in kidney examinations. We evaluated renal allograft stiffness using ARFI quantification in patients with stable renal function (n = 52) and those with biopsy-proven allograft dysfunction (n = 50). ARFI quantification, given as shear wave velocity (SWV), was performed. The resistance index (RI) was calculated by pulsed-wave Doppler ultrasound, and clinical and laboratory data were collected. Morphologic changes in transplanted kidneys were diagnosed by an independent pathologist. Mean SWV was more significantly negatively correlated with estimated glomerular filtration rate (eGFR) (r = -0.657, p < 0.0001) than was RI (r = -0.429, p = 0.0004) in transplanted kidneys. Receiver operating characteristic curve analysis revealed that the sensitivity and specificity of quantitative ultrasound in the diagnosis of renal allograft dysfunction were 72.0% and 86.5% (cutoff value = 2.625), respectively. The latter values were better than those of RI, which were 62.0% and 69.2% (cutoff value = 0.625), respectively. The coefficient of variation for repeat SWV measurements of the middle part of transplanted kidney was 8.64%, and inter-observer agreement on SWV was good (Bland-Altman method, ICC = 0.890). In conclusion, tissue elasticity quantification by ARFI is more accurate than the RI in diagnosing renal allograft function.

  5. 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.

  6. Polarized light spatial frequency domain imaging for non-destructive quantification of soft tissue fibrous structures.

    PubMed

    Yang, Bin; Lesicko, John; Sharma, Manu; Hill, Michael; Sacks, Michael S; Tunnell, James W

    2015-04-01

    The measurement of soft tissue fiber orientation is fundamental to pathophysiology and biomechanical function in a multitude of biomedical applications. However, many existing techniques for quantifying fiber structure rely on transmitted light, limiting general applicability and often requiring tissue processing. Herein, we present a novel wide-field reflectance-based imaging modality, which combines polarized light imaging (PLI) and spatial frequency domain imaging (SFDI) to rapidly quantify preferred fiber orientation on soft collagenous tissues. PLI utilizes the polarization dependent scattering property of fibers to determine preferred fiber orientation; SFDI imaging at high spatial frequency is introduced to reject the highly diffuse photons and to control imaging depth. As a result, photons scattered from the superficial layer of a multi-layered sample are highlighted. Thus, fiber orientation quantification can be achieved for the superficial layer with optical sectioning. We demonstrated on aortic heart valve leaflet that, at spatial frequency of f = 1mm(-1) , the diffuse background can be effectively rejected and the imaging depth can be limited, thus improving quantification accuracy. PMID:25909033

  7. Polarized light spatial frequency domain imaging for non-destructive quantification of soft tissue fibrous structures

    PubMed Central

    Yang, Bin; Lesicko, John; Sharma, Manu; Hill, Michael; Sacks, Michael S.; Tunnell, James W.

    2015-01-01

    The measurement of soft tissue fiber orientation is fundamental to pathophysiology and biomechanical function in a multitude of biomedical applications. However, many existing techniques for quantifying fiber structure rely on transmitted light, limiting general applicability and often requiring tissue processing. Herein, we present a novel wide-field reflectance-based imaging modality, which combines polarized light imaging (PLI) and spatial frequency domain imaging (SFDI) to rapidly quantify preferred fiber orientation on soft collagenous tissues. PLI utilizes the polarization dependent scattering property of fibers to determine preferred fiber orientation; SFDI imaging at high spatial frequency is introduced to reject the highly diffuse photons and to control imaging depth. As a result, photons scattered from the superficial layer of a multi-layered sample are highlighted. Thus, fiber orientation quantification can be achieved for the superficial layer with optical sectioning. We demonstrated on aortic heart valve leaflet that, at spatial frequency of f = 1mm−1, the diffuse background can be effectively rejected and the imaging depth can be limited, thus improving quantification accuracy. PMID:25909033

  8. [Temporal-spatial variations of reference evapotranspiration in Anhui Province and the quantification of the causes].

    PubMed

    Cao, Wen; Duan, Chun-feng; Yao, Yun; Yue, Wei

    2014-12-01

    In this paper, daily reference evapotranspiration (ET0) was computed with the recommended FAO-56 Penman-Monteith equation for Anhui Province using data collected 60 weather stations during 1961 to 2010 and its temporal-spatial variations were characterized. The determining factors in ET0 trends were inquired into through partial derivative quantification analysis for the study region. Results showed that the mean annual ET0 was 878.58 mm x a(-1) over the whole region during the study period. ET0 was the highest in summer and the lowest in winter. The mean annual ET0 decreased from the north to the south and from low altitude regions to high altitude regions. Both sunshine duration and wind speed were the dominant factors contributing to the interannual change of ET0, with less contribution from air temperature or relative humidity. The annual ET0 showed a general decline at a rate of -1.61 mm x a(-1) owing to a more negative contribution of sunshine duration and wind speed than a positive contribution of air temperature and relative humidity. ET0 increased insignificantly in spring and decreased slightly in both autumn and winter. However, it decreased significantly at a rate of -1.37 mm x a(-1) in summer. The main impacting factor was wind speed in spring, autumn and winter, but it was sunshine duration in summer. Great differences in the determining factors of the mean annual ET0 existed from area to area in Anhui Province. The wind speed was the determining factor for 36.7% of the whole stations distributing in the southern part of the area north to the Huaihe River and the area along the Huaihe River, while the sunshine duration was the determining factor for the other regions. PMID:25876416

  9. 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.

  10. 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.

  11. 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

  12. Reference Genes for Real-Time PCR Quantification of Messenger RNAs and MicroRNAs in Mouse Model of Obesity

    PubMed Central

    Matoušková, Petra; Bártíková, Hana; Boušová, Iva; Hanušová, Veronika; Szotáková, Barbora; Skálová, Lenka

    2014-01-01

    Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of NAD(P)H:quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice

  13. Rapid quantification of inflammation in tissue samples using perfluorocarbon emulsion and fluorine-19 nuclear magnetic resonance

    PubMed Central

    Ahrens, Eric T.; Young, Won-Bin; Xu, Hongyan; Pusateri, Lisa K.

    2016-01-01

    Quantification of inflammation in tissue samples can be a time-intensive bottleneck in therapeutic discovery and preclinical endeavors. We describe a versatile and rapid approach to quantitatively assay macrophage burden in intact tissue samples. Perfluorocarbon (PFC) emulsion is injected intravenously, and the emulsion droplets are effectively taken up by monocytes and macrophages. These ‘in situ’ labeled cells participate in inflammatory events in vivo resulting in PFC accumulation at inflammatory loci. Necropsied tissues or intact organs are subjected to conventional fluorine-19 (19F) NMR spectroscopy to quantify the total fluorine content per sample, proportional to the macrophage burden. We applied these methods to a rat model of experimental allergic encephalomyelitis (EAE) exhibiting extensive inflammation and demyelination in the central nervous system (CNS), particularly in the spinal cord. In a cohort of EAE rats, we used 19F NMR to derive an inflammation index (IFI) in intact CNS tissues. Immunohistochemistry was used to confirm intracellular colocalization of the PFC droplets within CNS CD68+ cells having macrophage morphology. The IFI linearly correlated to mRNA levels of CD68 via real-time PCR analysis. This 19F NMR approach can accelerate tissue analysis by at least an order of magnitude compared with histological approaches. PMID:21548906

  14. 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

  15. 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.

  16. 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.

  17. Extraction and Quantification of Carbon Nanotubes in Biological Matrices with Application to Rat Lung Tissue

    PubMed Central

    Doudrick, Kyle; Corson, Nancy; Oberdörster, Günter; Elder, Alison; Herckes, Pierre; Halden, Rolf U.; Westerhoff, Paul

    2013-01-01

    Extraction of carbon nanotubes (CNTs) from biological matrices such as rat lung tissue is integral to developing a quantification method for evaluating the environmental and human health exposure and toxicity of CNTs. The ability of various chemical treatment methods, including Solvable (2.5% sodium hydroxide/surfactant mixture), ammonium hydroxide, nitric acid, sulfuric acid, hydrochloric acid, hydrofluoric acid, hydrogen peroxide, and proteinase K, to extract CNTs from rat lung tissue was evaluated. CNTs were quantified using programmed thermal analysis (PTA). Two CNTs were used to represent the lower (500°C) and upper (800°C) PTA limit of CNT thermal stability. The recovery efficiency of each of the eight chemical reagents evaluated was found to depend on the ability to (1) minimize oxidation of CNTs, (2) remove interfering background carbon from the rat lung tissue, and (3) separate the solid-phase CNTs from the liquid-phase dissolved tissue via centrifugation. A two-step extraction method using Solvable and proteinase K emerged as the optimal approach, enabling a recovery of 98 ± 15% of a 2.9 ± 0.19 µg CNT loading that was spiked into whole rat lungs. Due to its high yield and applicability to low organ burdens of nanomaterials, this extraction method is particularly well suited for in vivo studies to quantify clearance rates and retained CNTs in lungs and other organs. PMID:23992048

  18. 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. PMID:24098735

  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. Use of cloned DNA fragments as reference materials for event specific quantification of genetically modified organisms (GMOs).

    PubMed

    Taverniers, I; Van Bockstaele, E; De Loose, M

    2001-01-01

    For the quantification of genetically modified organisms (GMOs) in foods and feeds, real-time PCR is currently the most widely applied technique. To obtain a % of GMO, a GMO-specific target sequence is quantified relatively to a species-specific sequence. The correctness and reliability of the obtained quantitative results fully depend on the reference materials used as standards for setting up external calibration curves. We introduced a completely new type of standards for quantification of GMOs, based on cloned plasmid DNA solutions with well-known amounts of the sequences of interest, expressed as copy numbers. Moreover, the junction sequence between inserted DNA and plant DNA was used as 'unique identifier'. In this study, the model was applied for Roundup Ready soybean.

  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. PMID:27396652

  3. 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

  4. 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.

  5. 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

  6. 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. PMID:26878463

  7. Reproducibility and observer variability of tissue phase mapping for the quantification of regional myocardial velocities.

    PubMed

    Lin, Kai; Chowdhary, Varun; Benzuly, Keith H; Yancy, Clyde W; Lomasney, Jon W; Rigolin, Vera H; Anderson, Allen S; Wilcox, Jane; Carr, James; Markl, Michael

    2016-08-01

    To systematically investigate the reproducibility of global and segmental left ventricular (LV) velocities derived from tissue phase mapping (TPM). Breath held and ECG synchronized TPM data (spatial/temporal resolution = 2 × 2 mm(2)/20.8 ms) were acquired in 18 healthy volunteers. To analyze scan-rescan variability, TPM was repeated in all subjects during a second visit separated by 16 ± 5 days. Data analysis included LV segmentation, and quantification of global and regional (AHA 16-segment modal) metrics of LV function [velocity-time curves, systolic and diastolic peak and time-to-peak (TTP) velocities] for radial (Vr), long-axis (Vz) and circumferential (VΦ) LV velocities. Mean velocity time curves in basal, mid-ventricular, and apical locations showed highly similar LV motion patterns for all three velocity components (Vr, VΦ, Vz) for scan and rescan. No significant differences for both systolic and diastolic peak and TTP myocardial velocities were observed. Segmental analysis revealed similar regional peak Vr and Vz during both systole and diastole except for three LV segments (p = 0.045, p = 0.033, and p = 0.009). Excellent (p < 0.001) correlations between scans and rescan for peak Vr (R(2) = 0.92), peak Vz (R(2) = 0.90), radial TTP (R(2) = 0.91) and long-axis TTP (R(2) = 0.88) confirmed good agreement. Bland-Altman analysis demonstrated excellent intra-observer and good inter-observer analysis agreement but increased variability for long axis peak velocities. TPM based analysis of global and regional myocardial velocities can be performed with good reproducibility. Robustness of regional quantification of long-axis velocities was limited but spatial velocity distributions across the LV could reliably be replicated. PMID:27116238

  8. 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.

  9. Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Anoectochilus roxburghii.

    PubMed

    Zhang, Gang; Zhao, Mingming; Song, Chao; Luo, Anxiong; Bai, Jianfa; Guo, Shunxing

    2012-05-01

    Accurate quantification of transcript profiling with quantitative real time polymerase chain reaction (qRT-PCR) relies on the reliable normalization of an appropriate reference gene. This study reported the identification and validation of nine reference genes, including β-tubulin (β-TUB), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), actin 1/2(ACT-1 and ACT-2), 18S rRNA, and 26S rRNA, from Anoectochilus roxburghii (Wall.) Lindl., a valuable herb remedy widely used for various diseases treatment in traditional Chinese medicine. Transcriptional levels of the candidate reference genes were examined using qRT-PCR analysis and revealed differential expression of the genes in the leaf, stem, root, flower, and peduncle tissues. The relative quantities data were subjected to geNorm software for ranking the expression stability of the reference genes and the results showed that EF-1β and ACT-2 were the two best stable genes whereas GAPDH and 26S rRNA did not favor normalization of qRT-PCR in these tissues. The expression pattern of a squalene synthase encoding gene (SS) was also determined in parallel. The analyses were in great consistency when the qRT-PCR data was normalized to the expression of each or both of EF-1β and ACT-2 as the internal control, further confirming the reliability of EF-1β and ACT-2 as the best internal control. The present study provided the first important clues for accurate data normalization in transcript profiling in A. roxburghii, which will be essential to further functional genomics study in the valuable medicinal plant. PMID:22201024

  10. Virtual Touch Tissue Imaging Quantification Shear Wave Elastography: Prospective Assessment of Cervical Lymph Nodes.

    PubMed

    Cheng, Kai Lun; Choi, Young Jun; Shim, Woo Hyun; Lee, Jeong Hyun; Baek, Jung Hwan

    2016-02-01

    The goal of this study was to prospectively evaluate the diagnostic performance of Virtual Touch tissue imaging quantification (VTIQ) shear wave elastography in the discrimination of benign and malignant cervical lymph nodes in routine clinical practice. Shear wave velocity was analyzed using VTIQ in 100 patients with 100 histologically proven cervical lymph nodes. Diagnostic performance was evaluated using receiver operating characteristic curve analysis and leave-one-out cross-validation. Agreement between measurements was assessed with intra-class correlation coefficients. The mean shear wave velocity was significantly higher in metastatic lymphadenopathy (4.46 ± 1.46 m/s) than in benign lymphadenopathy (2.71 ± 0.85 m/s) (p < 0.001) at a cutoff level of 3.34 m/s. The cross-validated accuracy, sensitivity and specificity were 77%, 78.9% and 74.4%, respectively. Agreement of measurements with VTIQ was excellent (intra-class correlation coefficient = 0.961). VTIQ shear wave elastography may be a feasible quantitative imaging method for differentiating benign and malignant cervical lymph nodes.

  11. Diagnosis of thyroid nodules using virtual touch tissue quantification value and anteroposterior/transverse diameter ratio.

    PubMed

    Li, Tao; Zhou, Pei; Zhang, Xuming; Ding, Mingyue; Yuchi, Ming; Li, Yiyong

    2015-02-01

    This paper proposes an effective approach to differential diagnosis of thyroid nodules using a hierarchical classification model based on the Virtual Touch tissue quantification (VTQ) value and anteroposterior/transverse diameter (A/T) ratio. One hundred twenty nodules (92 benign, 28 malignant) were analyzed using this approach by combining the quantitative elastic characteristic with the conventional sonographic feature. First, nodules were classified as benign (VTQ values <2.27 m/s), malignant (VTQ values >2.73 m/s) and indeterminate (2.27 m/s ≤ VTQ values ≤2.73 m/s) using two cutoff points selected on the basis of receiver operating characteristic analysis. Second, the indeterminate nodules were separated into malignant and benign nodules using an A/T ratio ≥1. The advantage of this approach was that it could alleviate the limitation of an overlap in VTQ values between benign and malignant nodules. According to the pathologic results, the accuracy of this approach was 95%. The proposed approach may potentially improve diagnostic accuracy.

  12. Quantification of tissue shrinkage in canine small intestinal specimens after resection and fixation

    PubMed Central

    Clarke, Ben S.; Banks, Tania A.; Findji, Laurent

    2014-01-01

    The aim of this study was to quantify the longitudinal shrinkage of canine small intestinal specimens after resection and fixation in 10% formalin. Samples were obtained from 12 clinically normal dogs of medium to large breed via ventral midline coeliotomy and enterectomy. The length of each sample was measured before excision, immediately after excision, and after 24 h in 10% formalin. The results were interpreted with the use of single-sample t-tests of the average changes; P-values of less than 0.01 were considered significant. The samples indicated a significant decrease in length after resection and fixation. The mean shrinkage from the pre-excision state was 28.3% immediately after excision (P < 0.0001) and 26.3% after 24 h of fixation (P < 0.0001). There was a small but not significant increase in the length of the specimens between the 2nd and 3rd measurement points. Quantification of the longitudinal shrinkage of resected intestinal specimens may improve interpretation of the distance of surgical margins from abnormal tissue in histopathology reports and allow investigation of the margins required for the clearance of specific tumors. PMID:24396180

  13. 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

  14. Quantification of the mRNA expression of G protein-coupled receptors in human adipose tissue.

    PubMed

    Amisten, Stefan

    2016-01-01

    G protein-coupled receptors (GPCRs) are important regulators of human physiology and therefore the targets of a large number of modern therapeutics. Although GPCRs are important regulators of adipose tissue endocrine and energy storage functions, the expression and function of a majority of GPCRs in adipose tissue is poorly characterized. A first step in the functional characterization of adipose tissue GPCRs is to accurately quantify the expression of GPCRs in adipose tissue. In this methods chapter, a detailed, step-by-step protocol is presented for the isolation of adipose tissue total RNA, its conversion into cDNA and the real-time PCR quantification of human GPCR mRNA expression relative to the mRNA expression of the stable adipose tissue housekeeping gene peptidylprolyl isomerase A (PPIA). A comprehensive list of 377 manually validated, commercially available GPCR qPCR primers allows facilitated swift quantification of either the entire human GPCRome or individual GPCRs, thus providing a sensitive, flexible, and cost-effective means of determining the mRNA expression of GPCRs in adipose tissue. PMID:26928540

  15. Quantification of myocardial salvage by myocardial perfusion SPECT and cardiac magnetic resonance--reference standards for ECG development.

    PubMed

    Engblom, Henrik; Aletras, Anthony H; Heiberg, Einar; Arheden, Håkan; Carlsson, Marcus

    2014-01-01

    In order to determine the cardioprotective efficacy of acute reperfusion therapy, assessed as myocardial salvage, in patients with acute coronary occlusion, the final myocardial infarct (MI) size needs to be related to the amount of ischemic myocardium during coronary occlusion, referred to as the myocardium at risk (MaR). There are currently several imaging approaches available for quantification of both MI size and MaR in vivo of which some have been validated both in pre-clinical and clinical settings. These methods often involve the use of either myocardial perfusion SPECT or cardiac magnetic resonance (CMR). These imaging methods could potentially be used to further develop and validate ECG methods for determination of MI size and MaR. Therefore, the aim of the present review is to give an overview of myocardial perfusion SPECT and CMR methods available for assessment of myocardial salvage by determination of MI size and MaR.

  16. Multiscale quantification of tissue spiculation and distortion for detection of architectural distortion and spiculated mass in mammography

    NASA Astrophysics Data System (ADS)

    Lao, Zhiqiang; Zheng, Xin

    2011-03-01

    This paper proposes a multiscale method to quantify tissue spiculation and distortion in mammography CAD systems that aims at improving the sensitivity in detecting architectural distortion and spiculated mass. This approach addresses the difficulty of predetermining the neighborhood size for feature extraction in characterizing lesions demonstrating spiculated mass/architectural distortion that may appear in different sizes. The quantification is based on the recognition of tissue spiculation and distortion pattern using multiscale first-order phase portrait model in texture orientation field generated by Gabor filter bank. A feature map is generated based on the multiscale quantification for each mammogram and two features are then extracted from the feature map. These two features will be combined with other mass features to provide enhanced discriminate ability in detecting lesions demonstrating spiculated mass and architectural distortion. The efficiency and efficacy of the proposed method are demonstrated with results obtained by applying the method to over 500 cancer cases and over 1000 normal cases.

  17. Development of green onion and cabbage certified reference materials for quantification of organophosphorus and pyrethroid pesticides.

    PubMed

    Otake, Takamitsu; Yarita, Takashi; Aoyagi, Yoshie; Kuroda, Youko; Numata, Masahiko; Iwata, Hitoshi; Mizukoshi, Kazushi; Nakamura, Munetomo; Watai, Masatoshi; Mitsuda, Hitoshi; Fujikawa, Takashi; Ota, Hidekazu

    2011-08-24

    Green onion and cabbage certified reference materials for the analysis of pesticide residues were issued by the National Metrology Institute of Japan, part of the National Institute of Advanced Industrial Science and Technology. Green onion and cabbage samples were grown so as to contain several kinds of organophosphorus and pyrethroid pesticides, and those were collected from a field in the Kochi Prefecture in Japan. The certification was carried out by using multiple analytical methods to ensure the reliability of analytical results; the values of target pesticides (diazinon, fenitrothion, cypermethrin, etofenprox, and permethrin for green onion and chlorpyrifos, fenitrothion, and permethrin for cabbage) were obtained by isotope dilution mass spectrometry. Certified values of target pesticides were 0.96-13.9 and 2.41-6.9 mg/kg for green onion and cabbage, respectively. These are the first green onion and cabbage powder certified reference materials in which organophosphorus and pyrethroid pesticides are determined. PMID:21774469

  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. Quantification of Gram-positive bacteria: adaptation and evaluation of a preparation strategy using high amounts of clinical tissue

    PubMed Central

    2014-01-01

    Background A preparation method for quantification of bacteria in tissues is obligatory to reduce tissue mass, concentrate the target, purify, remove inhibitory substances and to achieve constant target recovery rates. No preparation method has been available until now for a high mass of tissue applicable for routine use and analytical veterinary diagnostics. Results This study describes an easy-to-use tissue preparation protocol to quantify Gram-positive bacteria from a large volume of tissue matrix. A previously published sample preparation method (Matrix-Lysis) from food science was successfully adapted for clinical use on tissues from pigs, including cerebrum, spinal cord, lung, liver, ileum, colon, caecum, kidney and muscle tissue. This tissue preparation method now permits quantification of pathogens from 5 g of organic matrix, which is a 20–200 fold increase by weight compared to other methods. It is based on solubilization of the sample matrix with either a chaotrope plus detergent or divalent salts as solubilization agents. The method was designed as a modular system, offering the possibility to change lysis buffers, according to tissue solubilization characteristics and the intended detection method (molecular or culture). Using Listeria monocytogenes as model organism, viable cell quantification or DNA extraction and quantitative real-time PCR were performed after Matrix-Lysis to determine recovery rates and detection limit (LOD). The adapted Matrix-Lysis protocol resulted in high recovery rates (mean value: 76% ± 39%) for all tested organs, except kidney, and recovery was constant over 5 log scales for all tested buffer systems. The LOD for Matrix-Lysis with subsequent plate count method (PCM) was as low as 1 CFU/5 g, while for qPCR based detection the LOD was 102 bacterial cell equivalents (BCE)/5 g for two buffer systems. Conclusions This tissue preparation is inexpensive and can be easily used for routine and analytical veterinary

  20. Reference genes for real-time PCR quantification of microRNAs and messenger RNAs in rat models of hepatotoxicity.

    PubMed

    Lardizábal, María N; Nocito, Ana L; Daniele, Stella M; Ornella, Leonardo A; Palatnik, Javier F; Veggi, Luis M

    2012-01-01

    Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity.

  1. 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. PMID:25637570

  2. 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.

  3. Development of apple certified reference material for quantification of organophosphorus and pyrethroid pesticides.

    PubMed

    Otake, Takamitsu; Yarita, Takashi; Aoyagi, Yoshie; Kuroda, Youko; Numata, Masahiko; Iwata, Hitoshi; Watai, Masatoshi; Mitsuda, Hitoshi; Fujikawa, Takashi; Ota, Hidekazu

    2013-06-01

    An apple certified reference material for the analysis of pesticide residues was issued by the National Metrology Institute of Japan. Organophosphorus and pyrethroid pesticides were sprayed on apples, and these were used as raw materials of certified reference material. The harvested apples were cut into small pieces, freeze-dried, pulverized, sieved, placed into 200 brown glass bottles (3g each), and sterilized by γ-irradiation. Stability and homogeneity assessment was performed, and the relative uncertainties due to instability (for an expiry date of 32 months) and inhomogeneity were 10.3-25.0% and 4.0-6.8%, respectively. The characterization was carried out using multiple analytical methods to ensure the reliability of analytical results; the values of target pesticides were obtained by isotope dilution mass spectrometry. Certified values were 2.28 ± 0.82 mg/kg for diazinon, 3.14 ± 0.79 mg/kg for fenitrothion, 1.55 ± 0.81 mg/kg for cypermethrin, and 2.81 ± 0.70 mg/kg for permethrin.

  4. 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

  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. 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

  8. 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.

  9. Automated segmentation of reference tissue for prostate cancer localization in dynamic contrast enhanced MRI

    NASA Astrophysics Data System (ADS)

    Vos, Pieter C.; Hambrock, Thomas; Barentsz, Jelle O.; Huisman, Henkjan J.

    2010-03-01

    For pharmacokinetic (PK) analysis of Dynamic Contrast Enhanced (DCE) MRI the arterial input function needs to be estimated. Previously, we demonstrated that PK parameters have a significant better discriminative performance when per patient reference tissue was used, but required manual annotation of reference tissue. In this study we propose a fully automated reference tissue segmentation method that tackles this limitation. The method was tested with our Computer Aided Diagnosis (CADx) system to study the effect on the discriminating performance for differentiating prostate cancer from benign areas in the peripheral zone (PZ). The proposed method automatically segments normal PZ tissue from DCE derived data. First, the bladder is segmented in the start-to-enhance map using the Otsu histogram threshold selection method. Second, the prostate is detected by applying a multi-scale Hessian filter to the relative enhancement map. Third, normal PZ tissue was segmented by threshold and morphological operators. The resulting segmentation was used as reference tissue to estimate the PK parameters. In 39 consecutive patients carcinoma, benign and normal tissue were annotated on MR images by a radiologist and a researcher using whole mount step-section histopathology as reference. PK parameters were computed for each ROI. Features were extracted from the set of ROIs using percentiles to train a support vector machine that was used as classifier. Prospective performance was estimated by means of leave-one-patient-out cross validation. A bootstrap resampling approach with 10,000 iterations was used for estimating the bootstrap mean AUCs and 95% confidence intervals. In total 42 malignant, 29 benign and 37 normal regions were annotated. For all patients, normal PZ was successfully segmented. The diagnostic accuracy obtained for differentiating malignant from benign lesions using a conventional general patient plasma profile showed an accuracy of 0.64 (0.53-0.74). Using the

  10. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material

    PubMed Central

    Dong, Lianhua; Meng, Ying; Sui, Zhiwei; Wang, Jing; Wu, Liqing; Fu, Boqiang

    2015-01-01

    Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments. PMID:26302947

  11. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.

    PubMed

    Dong, Lianhua; Meng, Ying; Sui, Zhiwei; Wang, Jing; Wu, Liqing; Fu, Boqiang

    2015-01-01

    Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments.

  12. 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.

  13. 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. PMID:23052591

  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. Semi-automated quantification of axonal densities in labeled CNS tissue.

    PubMed

    Grider, Michael H; Chen, Qin; Shine, H David

    2006-09-15

    Current techniques used to quantify axons often rely upon manual quantification or potentially expensive commercially available programs for automated quantification. We describe a computerized method for the detection and quantification of axons in the rat CNS using readily available free software. Feature J, a java-based plug-in to the imaging software NIH Image J, faithfully detects linear structures such as axons in confocal or bright-field images using a Hessian-based algorithm. We validated the method by comparing values obtained by manual and automated analyses of axons induced to grow in response to neurotrophin over-expression in the rat spinal cord. We also demonstrated that the program can be used to quantify neurotrophin-induced growth of lesioned serotonergic axons in the rat cortex, where manual measurement would be impractical due to dense axonal growth. The use of this software suite provided faster and less biased quantification of labeled axons in comparison to manual measurements at no cost.

  16. 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

  17. Overcoming stability challenges in the quantification of tissue nucleotides: determination of 2'-C-methylguanosine triphosphate concentration in mouse liver.

    PubMed

    Rashidzadeh, Hassan; Bhadresa, Sanjeev; Good, Steven Spencer; Larsson Cohen, Marita; Gupta, Kusum Sachdev; Rush, William Roger

    2015-01-01

    A conventional, rapid and high throughput method for tissue extraction and accurate and selective LC-MS/MS quantification of 2'-C-methylguanosine triphosphate (2'-MeGTP) in mouse liver was developed and qualified. Trichloroacetic acid (TCA) was used as the tissue homogenization reagent that overcomes instability challenges of liver tissue nucleotide triphosphates due to instant ischemic degradation to mono- and diphosphate nucleotides. Degradation of 2'-MeGTP was also minimized by harvesting livers using in situ clamp-freezing or snap-freezing techniques. The assay also included a sample clean-up procedure using weak anion exchange solid phase extraction followed by ion exchange chromatography and tandem mass spectrometry detection. The linear assay range was from 50 to 10000 pmol/mL concentration in liver homogenate (250-50000 pmol/g in liver tissue). The method was qualified over three intraday batches for accuracy, precision, selectivity and specificity. The assay was successfully applied to pharmacokinetic studies of 2'-MeGTP in liver tissue samples after single oral doses of IDX184, a nucleotide prodrug inhibitor of the viral polymerase for the treatment of hepatitis C, to mice. The study results suggested that the clamp-freezing liver collection method was marginally more effective in preventing 2'-MeGTP degradation during liver tissue collection compared to the snap-freezing method. PMID:25757919

  18. 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.

  19. Tissue-specific transcriptome sequencing analysis expands the non-human primate reference transcriptome resource (NHPRTR)

    PubMed Central

    Peng, Xinxia; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Nishida, Andrew; Pipes, Lenore; Bozinoski, Marjan; Thomas, Matthew J.; Kelly, Sara; Weiss, Jeffrey M.; Raveendran, Muthuswamy; Muzny, Donna; Gibbs, Richard A.; Rogers, Jeffrey; Schroth, Gary P.; Katze, Michael G.; Mason, Christopher E.

    2015-01-01

    The non-human primate reference transcriptome resource (NHPRTR, available online at http://nhprtr.org/) aims to generate comprehensive RNA-seq data from a wide variety of non-human primates (NHPs), from lemurs to hominids. In the 2012 Phase I of the NHPRTR project, 19 billion fragments or 3.8 terabases of transcriptome sequences were collected from pools of ∼20 tissues in 15 species and subspecies. Here we describe a major expansion of NHPRTR by adding 10.1 billion fragments of tissue-specific RNA-seq data. For this effort, we selected 11 of the original 15 NHP species and subspecies and constructed total RNA libraries for the same ∼15 tissues in each. The sequence quality is such that 88% of the reads align to human reference sequences, allowing us to compute the full list of expression abundance across all tissues for each species, using the reads mapped to human genes. This update also includes improved transcript annotations derived from RNA-seq data for rhesus and cynomolgus macaques, two of the most commonly used NHP models and additional RNA-seq data compiled from related projects. Together, these comprehensive reference transcriptomes from multiple primates serve as a valuable community resource for genome annotation, gene dynamics and comparative functional analysis. PMID:25392405

  20. Selection and Evaluation of Tissue Specific Reference Genes in Lucilia sericata during an Immune Challenge

    PubMed Central

    Baumann, Andre; Lehmann, Rüdiger; Beckert, Annika; Vilcinskas, Andreas; Franta, Zdeněk

    2015-01-01

    The larvae of the common green bottle fly Lucilia sericata (Diptera: Calliphoridae) have been used for centuries to promote wound healing, but the molecular basis of their antimicrobial, debridement and healing functions remains largely unknown. The analysis of differential gene expression in specific larval tissues before and after immune challenge could be used to identify key molecular factors, but the most sensitive and reproducible method qRT-PCR requires validated reference genes. We therefore selected 10 candidate reference genes encoding products from different functional classes (18S rRNA, 28S rRNA, actin, β-tubulin, RPS3, RPLP0, EF1α, PKA, GAPDH and GST1). Two widely applied algorithms (GeNorm and Normfinder) were used to analyze reference gene candidates in different larval tissues associated with secretion, digestion, and antimicrobial activity (midgut, hindgut, salivary glands, crop and fat body). The Gram-negative bacterium Pseudomonas aeruginosa was then used to boost the larval immune system and the stability of reference gene expression was tested in comparison to three immune genes (lucimycin, defensin-1 and attacin-2), which target different pathogen classes. We observed no differential expression of the antifungal peptide lucimycin, whereas the representative targeting Gram-positive bacteria (defensin-1) was upregulated in salivary glands, crop, nerve ganglion and reached its maximum in fat body (up to 300-fold). The strongest upregulation in all immune challenged tissues (over 50,000-fold induction in the fat body) was monitored for attacin-2, the representative targeting Gram-negative bacteria. Here we identified and validated a set of reference genes that allows the accurate normalization of gene expression in specific tissues of L. sericata after immune challenge. PMID:26252388

  1. Selection and Evaluation of Tissue Specific Reference Genes in Lucilia sericata during an Immune Challenge.

    PubMed

    Baumann, Andre; Lehmann, Rüdiger; Beckert, Annika; Vilcinskas, Andreas; Franta, Zdeněk

    2015-01-01

    The larvae of the common green bottle fly Lucilia sericata (Diptera: Calliphoridae) have been used for centuries to promote wound healing, but the molecular basis of their antimicrobial, debridement and healing functions remains largely unknown. The analysis of differential gene expression in specific larval tissues before and after immune challenge could be used to identify key molecular factors, but the most sensitive and reproducible method qRT-PCR requires validated reference genes. We therefore selected 10 candidate reference genes encoding products from different functional classes (18S rRNA, 28S rRNA, actin, β-tubulin, RPS3, RPLP0, EF1α, PKA, GAPDH and GST1). Two widely applied algorithms (GeNorm and Normfinder) were used to analyze reference gene candidates in different larval tissues associated with secretion, digestion, and antimicrobial activity (midgut, hindgut, salivary glands, crop and fat body). The Gram-negative bacterium Pseudomonas aeruginosa was then used to boost the larval immune system and the stability of reference gene expression was tested in comparison to three immune genes (lucimycin, defensin-1 and attacin-2), which target different pathogen classes. We observed no differential expression of the antifungal peptide lucimycin, whereas the representative targeting Gram-positive bacteria (defensin-1) was upregulated in salivary glands, crop, nerve ganglion and reached its maximum in fat body (up to 300-fold). The strongest upregulation in all immune challenged tissues (over 50,000-fold induction in the fat body) was monitored for attacin-2, the representative targeting Gram-negative bacteria. Here we identified and validated a set of reference genes that allows the accurate normalization of gene expression in specific tissues of L. sericata after immune challenge.

  2. The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue.

    PubMed

    Dowling, Catríona M; Walsh, Dara; Coffey, John C; Kiely, Patrick A

    2016-01-01

    Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) remains the most sensitive technique for nucleic acid quantification. Its popularity is reflected in the remarkable number of publications reporting RT-qPCR data. Careful normalisation within RT-qPCR studies is imperative to ensure accurate quantification of mRNA levels. This is commonly achieved through the use of reference genes as an internal control to normalise the mRNA levels between different samples. The selection of appropriate reference genes can be a challenge as transcript levels vary with physiology, pathology and development, making the information within the transcriptome flexible and variable. In this study, we examined the variation in expression of a panel of nine candidate reference genes in HCT116 and HT29 2-dimensional and 3-dimensional cultures, as well as in normal and cancerous colon tissue. Using normfinder we identified the top three most stable genes for all conditions. Further to this we compared the change in expression of a selection of PKC coding genes when the data was normalised to one reference gene and three reference genes. Here we demonstrated that there is a variation in the fold changes obtained dependent on the number of reference genes used. As well as this, we highlight important considerations namely; assay efficiency tests, inhibition tests and RNA assessment which should also be implemented into all RT-qPCR studies. All this data combined demonstrates the need for careful experimental design in RT-qPCR studies to help eliminate false interpretation and reporting of results.

  3. 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.

  4. 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. PMID:26451284

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

    PubMed Central

    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. PMID:26451284

  6. 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.

  7. Acoustic radiation force impulse imaging with virtual touch tissue quantification: measurements of normal breast tissue and dependence on the degree of pre-compression.

    PubMed

    Wojcinski, Sebastian; Brandhorst, Kathrin; Sadigh, Gelareh; Hillemanns, Peter; Degenhardt, Friedrich

    2013-12-01

    Acoustic radiation force impulse imaging (ARFI) with Virtual Touch tissue quantification (VTTQ) enables the determination of shear wave velocity in meters per second (m/s). We investigated shear wave velocity in normal breast tissue and analyzed the influence of the degree of pre-compression on the measurements. In repeated measurements and with normal pre-compression, the mean shear wave velocity in breast parenchyma was significantly higher than that in breast adipose tissue (3.33 ± 1.18 m/s vs. 2.90 ± 1.10 m/s; p < 0.001; 712 measurements in 89 patients). Furthermore, we found a significant positive correlation between degree of pre-compression and velocity measurements. Shear wave velocities with low, moderate and high pre-compression were 1.89, 3.18 and 4.39 m/s in parenchyma, compared with 1.46, 2.55 and 3.64 m/s in adipose tissue, respectively (p < 0.001; 360 measurements in 60 patients). VTTQ of breast tissue is a feasible method with high accuracy; however, the degree of pre-compression applied may significantly influence the measurements.

  8. A Preliminary Investigation of Normal Pancreas and Acute Pancreatitis Elasticity Using Virtual Touch Tissue Quantification (VTQ) Imaging

    PubMed Central

    Xie, Juan; Zou, Liling; Yao, Minghua; Xu, Guang; Zhao, Lixia; Xu, Huixiong; Wu, Rong

    2015-01-01

    Background The aim of this study was to prospectively evaluate the use of elastometry in healthy volunteers and patients with acute pancreatitis using virtual touch tissue quantification (VTQ) imaging technology performed on the pancreas. Material/Methods We enrolled 210 healthy volunteers and 44 acute pancreatitis patients in the study between March 2012 and June 2013. Healthy subjects were divided into 3 groups: young (18–30 years), middle-aged (30–50 years), and elderly (>50 years). VTQ was performed on the pancreatic head and body regions to obtain shear wave velocity (SWV) measurements, which were used to evaluate the elasticity values of tissues. Results The pancreatic head SWV value in the whole healthy group was 1.18±0.23 m/s, and that in the pancreatic body was 1.21±0.20 m/s. In patients with acute pancreatitis, the mean SWV measurements at the head were 1.18±0.20 m/s, compared to 1.25±0.19 m/s in the pancreatic body. There was no statistically significant difference between whole healthy volunteers and the acute pancreatitis group. Conclusions VTQ is a new method that shows promise for the quantification of pancreatic elasticity, but further studies are warranted. PMID:26062803

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

    PubMed

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

    2013-12-01

    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

  10. 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

  11. 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.

  12. 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.

  13. 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

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

    PubMed

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

    2016-08-30

    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.

  15. 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

  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. 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.

  18. Investigating reference genes for quantitative real-time PCR analysis across four chicken tissues.

    PubMed

    Bagés, S; Estany, J; Tor, M; Pena, R N

    2015-04-25

    Accurate normalization of data is required to correct for different efficiencies and errors during the processing of samples in reverse transcription PCR analysis. The chicken is one of the main livestock species and its genome was one of the first reported and used in large scale transcriptomic analysis. Despite this, the chicken has not been investigated regarding the identification of reference genes suitable for the quantitative PCR analysis of growth and fattening genes. In this study, five candidate reference genes (B2M, RPL32, SDHA, TBP and YWHAZ) were evaluated to determine the most stable internal reference for quantitative PCR normalization in the two main commercial muscles (pectoralis major (breast) and biceps femoris (thigh)), liver and abdominal fat. Four statistical methods (geNorm, NormFinder, CV and BestKeeper) were used in the evaluation of the most suitable combination of reference genes. Additionally, a comprehensive ranking was established with the RefFinder tool. This analysis identified YWHAZ and TBP as the recommended combination for the analysis of biceps femoris and liver, YWHAZ and RPL32 for pectoralis major and RPL32 and B2M for abdominal fat and across-tissue studies. The final ranking for each tool changed slightly but overall the results, and most particularly the ability to discard the least robust candidates, were consistent between tools. The selection and number of reference genes were validated using SCD, a target gene related to fat metabolism. Overall, the results can be directly used to quantitate target gene expression in different tissues or in validation studies from larger transcriptomic experiments.

  19. 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.

  20. 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.

  1. 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. PMID:26968570

  2. Quantification of extracellular matrix proteins from a rat lung scaffold to provide a molecular readout for tissue engineering.

    PubMed

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

    2015-04-01

    The use of extracellular matrix (ECM) 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.

  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. 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.

  5. 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

    2016-02-01

    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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Chromophore absorbance change quantification in tissue during low-level light therapy

    NASA Astrophysics Data System (ADS)

    Huynh, Daniel; Chung, Christine; Qian, Li; Lilge, Lothar

    2012-03-01

    Low Level Light Therapy (LLLT) has been implicated to stimulate tissue, promoting healing and reducing pain. One of the potential pathways stimulated by LLLT relates to the electron transport chain, where photon quantum energy can induce a change in the biochemical reactions within the cell. The aim of this study is to assess the feasibility to exploit light additionally as a diagnostic tool to determine tissue physiological states, particularly in quantifying the changes in redox states of Cytochrome C as a result of induced LLLT biochemical reactions.

  12. Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach

    NASA Astrophysics Data System (ADS)

    Xu, Xiaochun; Sinha, Lagnojita; Singh, Aparna; Yang, Cynthia; Xiang, Jialing; Tichauer, Kenneth M.

    2015-03-01

    Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, "untargeted" imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.

  13. 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...

  14. An Integrated Indenter-ARFI Imaging System for Tissue Stiffness Quantification

    PubMed Central

    Zhai, Liang; Palmeri, Mark L.; Bouchard, Richard R.; Nightingale, Roger W.; Nightingale, Kathryn R.

    2008-01-01

    The goal of this work is to develop and characterize an integrated indenter-ARFI (acoustic radiation force impulse) imaging system. This system is capable of acquiring matched datasets of ARFI images and stiffness profiles from ex vivo tissue samples, which will facilitate correlation of ARFI images of tissue samples with independently-characterized material properties. For large and homogeneous samples, the indenter can be used to measure the Young's moduli by using Boussinesq's solution for a load on the surface of a semi-infinite isotropic elastic medium. Experiments and finite element method (FEM) models were designed to determine the maximum indentation depth and minimum sample size for accurate modulus reconstruction using this solution. Applying these findings, indentation measurements were performed on three calibrated commercial tissue-mimicking phantoms and the results were in good agreement with the calibrated stiffness. For heterogeneous tissue samples, indentation can be used independently to characterize relative stiffness variation across the sample surface, which can then be used to validate the stiffness variation in registered ARFI images. Tests were performed on heterogeneous phantoms and freshly-excised colon cancer specimens to detect the relative stiffness and lesion sizes using the combined system. Normalized displacement curves across the lesion surface were calculated and compared. Good agreement of the lesion profiles was observed between indentation and ARFI imaging. PMID:18939611

  15. 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.

  16. 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.

  17. Quantification of the tissue-culture induced variation in barley (Hordeum vulgare L.)

    PubMed Central

    Bednarek, Piotr T; Orłowska, Renata; Koebner, Robert MD; Zimny, Janusz

    2007-01-01

    Background When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level. Results A variant of methylation sensitive AFLP, based on the isoschizomeric combinations Acc65I/MseI and KpnI/MseI was applied to analyze, at both the sequence and methylation levels, the outcomes of regeneration from tissue culture in barley. Both sequence mutation and alteration in methylation pattern were detected. Two sets of regenerants from each of five DH donor lines were compared. One set was derived via androgenesis, and the other via somatic embryogenesis, developed from immature embryos. These comparisons delivered a quantitative assessment of the various types of somaclonal variation induced. The average level of variation was 6%, of which almost 1.7% could be accounted for by nucleotide mutation, and the remainder by changes in methylation state. The nucleotide mutation rates and the rate of epimutations were substantially similar between the andro- and embryo-derived sets of regenerants across all the donors. Conclusion We have developed an AFLP based approach that is capable of describing the qualitative and quantitative characteristics of the tissue culture-induced variation. We believe that this approach will find particular value in the study of patterns of inheritance of somaclonal variation, since non-heritable variation is of little interest for the improvement of plant species which are sexually

  18. Absolute quantification of a very virulent Marek's disease virus dynamic quantity and distributions in different tissues.

    PubMed

    Zhang, Zhenjie; Liu, Shaoqiong; Ma, Chengtai; Zhao, Peng; Cui, Zhizhong

    2015-06-01

    Chickens infected with Marek's disease virus (MDV) carry the virus consistently for a long time, which increases the incidence and rate of virus-induced multi-organ tumors and increases its potential for horizontal transmission. There is a positive correlation between very virulent (vv) MDV quantity and the pathology. The purpose of this study was to determine the vvMDV loads dynamics in different phases, and the correlation between the viral quantity and tumor development. We used a SYBR Green duplex real-time quantitative PCR (q-PCR) assay to detect and quantify MDV loads and distributions in different tissues, targeting the Eco-Q protein gene (meq) of the virus and the house-keeping ovotransferrin (ovo) gene of chickens. The q-PCR was performed using different tissue DNA preparations derived from chickens which were infected with 1,000 pfu of the SDWJ1302 strain and tissue samples were collected from control and MDV-infected birds on 7, 10, 15, 21, 28, 40, 60, and 90 d post-infection (DPI). The data indicated that the MDV genome was almost quantifiable in immune organs of infected chickens as early as 7 DPI, and the number of MDV genome copies in the blood and different organs peaked by 28 DPI, but then gradually decreased by 40 DPI. The levels of viral quantity in the lymphocytes, liver, and spleen were all higher than those in other organs, and that in the feather follicles was the highest among different phases of MDV infection. The vvMDV could still be detected in peripheral blood and tissues by 90 DPI, and the vast existence of virus will stimulate tissue destruction. The data provided further evidence of viral infection involving multi-organ distribution and mainly involving immune organ proliferation, resulting in immunosuppression. PMID:25834249

  19. Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development.

    PubMed

    Hirashima, Tsuyoshi; Adachi, Taiji

    2015-01-01

    Whole-tissue quantification at single-cell resolution has become an inevitable approach for further quantitative understanding of morphogenesis in organ development. The feasibility of the approach has been dramatically increased by recent technological improvements in optical tissue clearing and microscopy. However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed. To provide the appropriate procedure, we here show tips for each critical step of the entire process, including fixation for immunofluorescence, optical clearing, and digital image processing, using developing murine internal organs such as epididymis, kidney, and lung as an example. Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging. In addition, we demonstrated that three-dimensional digital image processing after optical clearing produces objective quantitative data for the whole-tissue analysis, focusing on the spatial distribution of mitotic cells in the epididymal tubule. The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level. PMID:26258587

  20. 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. PMID:25736591

  1. 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.

  2. 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. PMID:27617230

  3. Quantification of fibrosis and mast cells in the tissue response of endodontic sealer irradiated by low-level laser therapy.

    PubMed

    Berbert, Fábio Luiz Camargo Villela; Sivieri-Araújo, Gustavo; Ramalho, Lizeti Toledo Oliveira; Pereira, Sanívia Aparecida Lima; Rodrigues, Denise Bertulucci Rocha; de Araújo, Marcelo Sivieri

    2011-11-01

    Low-level laser therapy (LLLT) accelerates tissue repair. Mast cells induce the proliferation of fibroblasts and the development of local fibrosis. The objective of this study was to quantify fibrosis rate and mast cells in connective tissue after endodontic sealer zinc oxide and eugenol (ZOE) was implanted and submitted to LLLT, immediately after implant and again 24 h later. Sixty mice were distributed into three groups: GI, GII, and GIII (n = 20). In GI, the tubes filled with Endofill were implanted in the animals and were not irradiated with LLLT. In GII, the tubes containing Endofill were implanted in the animals and then irradiated with red LLLT (InGaAIP) 685-nm wavelength, D = 72 J/Cm(2), E = 2 J, T = 58 s, P = 35 mW, and in GIII, the tubes with Endofill were implanted and irradiated with infrared LLLT (AsGaAl) 830-nm wavelength, D = 70 J/Cm(2), E = 2 J, T = 40 s, P = 50 mW. After 7 days and 30 days, the animals were killed. A series of 6-µm-thick sections were obtained and stained with Toluidine Blue and Picrosirius and analyzed under a standard light microscope using a polarized light filter for the quantification of fibrosis. The statistics were qualitative and quantitative with a significance of 5%. The irradiation with LLLT did not offer improvement in the fibrosis rate, however, it provided a significant decrease in the concentration of independent mast cells for the period studied.

  4. An improved simplified high-sensitivity quantification method for determining brassinosteroids in different tissues of rice and Arabidopsis.

    PubMed

    Xin, Peiyong; Yan, Jijun; Fan, Jinshi; Chu, Jinfang; Yan, Cunyu

    2013-08-01

    Quantification of brassinosteroids is essential and extremely important to study the molecular mechanisms of their physiological roles in plant growth and development. Herein, we present a simple, material and cost-saving high-performance method for determining endogenous brassinosteroids (BRs) in model plants. This new method enables simultaneous enrichment of a wide range of bioactive BRs such as brassinolide, castasterone, teasterone, and typhasterol with ion exchange solid-phase extraction and high-sensitivity quantitation of these BRs based on isotope dilution combined with internal standard approach. For routine analysis, the consumption of plant materials was reduced to one-twentieth of previously reported and the overall process could be completed within 1 day compared with previous 3 to 4 days. The strategy was validated by profiling BRs in different ecotypes and mutants of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the BR distributions in different model plants tissues were determined with the new method. The method allows plant physiologists to monitor the dynamics and distributions of BRs with 1 gram fresh weight of model plant tissues, which will speed up the process for the molecular mechanism research of BRs with these model plants in future work.

  5. 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.

  6. 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.

  7. 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.

  8. Quantification of human tissue transglutaminase by a luminescence sandwich enzyme-linked immunosorbent assay.

    PubMed

    Wolf, Johannes; Lachmann, Ingolf; Wagner, Uta; Osman, Awad A; Mothes, Thomas

    2011-12-15

    Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes crosslinking of peptidic glutamine residues with primary amines via isopeptide bonds and hydrolysis of ATP or GTP. The enzyme exerts a variety of functions at the cellular and tissue levels that may be disturbed in disease. Its role in pathoprocesses is poorly understood. For investigation of the involvement of tTG in disease, sensitive and specific assays should be available. We have developed the first sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mabs) against human tTG. tTG is captured by mab 3C10 and detected by biotinylated mab 10F3. After incubation with peroxidase-conjugated streptavidin, bound tTG is visualized by peroxidase reaction applying a luminescence substrate. The detection limit was 40 pg/ml. The assay was highly reproducible. Recovery of spiked tTG in crude samples was greater than 92%. The enzyme could be detected in cellular lysates and tissue homogenates of humans. The effect of typical effectors (retinoic acid and interferon-γ) on tTG expression could be demonstrated. A low signal was also obtained in mice samples, suggesting cross-reactivity of the mabs with murine tTG. The new sandwich ELISA may be successfully applied for investigation of physiological functions of tTG and of disorders associated with inadequate tTG expression.

  9. Simultaneous quantification of flow and tissue velocities based on multi-angle plane wave imaging.

    PubMed

    Ekroll, Ingvild Kinn; Swillens, Abigail; Segers, Patrick; Dahl, Torbjørn; Torp, Hans; Lovstakken, Lasse

    2013-04-01

    A quantitative angle-independent 2-D modality for flow and tissue imaging based on multi-angle plane wave acquisition was evaluated. Simulations of realistic flow in a carotid artery bifurcation were used to assess the accuracy of the vector Doppler (VD) technique. Reduction in root mean square deviation from 27 cm/s to 6 cm/s and 7 cm/s to 2 cm/s was found for the lateral (vx) and axial (vz) velocity components, respectively, when the ensemble size was increased from 8 to 50. Simulations of a Couette flow phantom (vmax = 2.7 cm/s) gave promising results for imaging of slowly moving tissue, with root mean square deviation of 4.4 mm/s and 1.6 mm/s for the x- and z-components, respectively. A packet acquisition scheme providing both B-mode and vector Doppler RF data was implemented on a research scanner, and beamforming and further post-processing was done offline. In vivo results of healthy volunteers were in accordance with simulations and gave promising results for flow and tissue vector velocity imaging. The technique was also tested in patients with carotid artery disease. Using the high ensemble vector Doppler technique, blood flow through stenoses and secondary flow patterns were better visualized than in ordinary color Doppler. Additionally, the full velocity spectrum could be obtained retrospectively for arbitrary points in the image.

  10. Drug Detection and Quantification Directly from Tissue using Novel Ionization Methods for Mass Spectrometry

    PubMed Central

    Wang, Beixi; Dearring, Chenelle L.; Wager-Miller, James; Mackie, Ken; Trimpin, Sarah

    2016-01-01

    Solvent assisted ionization inlet (SAII) and matrix assisted ionization vacuum (MAIV) were used to rapidly quantify an antipsychotic drug, clozapine, directly from surfaces with minimal sample preparation. This simple surface analysis method based on SAII- and MAIV-mass spectrometry (MS) was developed to allow detection of endogenous lipids, metabolites, and clozapine directly from mouse brain tissue sections. Rapid surface assessment was achieved by SAII with the assistance of heat on the mass spectrometer inlet, and MAIV showed capability on heat-limited instruments with better reproducibility. In addition, isotope dilution and standard addition were used without sample clean-up, and the results correlate well to liquid chromatography (LC)-tandem MS with sample work-up. Using the simple surface methods, standard solutions containing clozapine and deuterated sample (clozapine-d8) at different concentration ratios were used to extract and quantify clozapine from brain tissue sections of drug-treated mouse at different thicknesses. The amount of clozapine extracted by these surface methods was independent of tissue thickness. PMID:26307700

  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. Laser-induced breakdown spectroscopy: a new approach for nanoparticle's mapping and quantification in organ tissue.

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  14. Quantification of α-Gal Antigen Removal in the Porcine Dermal Tissue by α-Galactosidase.

    PubMed

    Gao, Hong-Wei; Li, Su-Bo; Sun, Wendell Q; Yun, Zhi-Min; Zhang, Xue; Song, Jin-Wen; Zhang, Shi-Kun; Leng, Ling; Ji, Shou-Ping; Tan, Ying-Xia; Gong, Feng

    2015-11-01

    The α-Gal (Galα1,3-Galβ1-4GlcNAc-R) epitope, the major xenoantigen, is the first barrier in a porcine-to-man tissue and organ xenotransplantation. The elimination or reduction of the α-Gal epitopes is therefore an important step for a successful xenotransplantation. The present study is to evaluate the α-Gal elimination in the porcine skin with α-galactosidase treatment, and to assess two methods (immunohistochemistry and inhibition ELISA) that may be used in quality control for quantifying the extent of the α-Gal elimination. Enzymatic cleavage in a single-step process is extremely efficient and affordable at eliminating the α-Gal epitope even in a tissue as dense as the porcine dermis. The cost of enzymatic cleavage is found to be less than US$7 for a 10 × 10 cm piece of porcine skin (0.5 mm thick) or about US$140 for 100 g of 3-dimensional soft tissues. After enzymatic cleavage, the α-Gal-positive immunostaining was essentially undetectable in enzyme-treated porcine skin. The inhibition rate constant of the monoclonal anti-Gal antibody M86 binding to α-Gal-bovine serum albumin in ELISA was reduced from 15.0 ± 4.3 (n = 10) to 6.1 ± 2.6 (n = 7) after enzyme treatment, in comparison to 4.4 ± 1.8 (n = 9) background inhibition of decellularized human skin (the ultimate negative control), which demonstrates ∼ 84% elimination of α-Gal epitopes in treated porcine skin. To examine the suitability of two detection methods for the routine quality control application, comparative studies were made with control and enzyme-treated porcine skin, porcine skin from the α-Gal knockout animal, as well as decellularized human skin. The data show that the traditional immunohistochemistry and, to a less extent, the inhibition ELISA with further modifications can be used as quality control tools in the production and selection of biocompatible bioprosthetic devices. The biological evaluation of enzyme-treated porcine skin is ongoing with a small animal model and a

  15. A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids.

    PubMed

    McCarron, Pearse; Giddings, Sabrina D; Reeves, Kelley L; Hess, Philipp; Quilliam, Michael A

    2015-04-01

    Azaspiracids (AZAs) are lipophilic biotoxins produced by marine algae that can contaminate shellfish and cause human illness. The European Union (EU) regulates the level of AZAs in shellfish destined for the commercial market, with liquid chromatography-mass spectrometry (LC-MS) being used as the official reference method for regulatory analysis. Certified reference materials (CRMs) are essential tools for the development, validation, and quality control of LC-MS methods. This paper describes the work that went into the planning, preparation, characterization, and certification of CRM-AZA-Mus, a tissue matrix CRM, which was prepared as a wet homogenate from mussels (Mytilus edulis) naturally contaminated with AZAs. The homogeneity and stability of CRM-AZA-Mus were evaluated, and the CRM was found to be fit for purpose. Extraction and LC-MS/MS methods were developed to accurately certify the concentrations of AZA1 (1.16 mg/kg), AZA2 (0.27 mg/kg), and AZA3 (0.21 mg/kg) in the CRM. Quantitation methods based on standard addition and matrix-matched calibration were used to compensate for the matrix effects in LC-MS/MS. Other toxins present in this CRM at lower levels were also measured with information values reported for okadaic acid, dinophysistoxin-2, yessotoxin, and several spirolides.

  16. 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.

  17. 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.

  18. 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. PMID:24771348

  19. High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications.

    PubMed

    Fridman, Yulia; Holland, Neta; Elbaum, Rivka; Savaldi-Goldstein, Sigal

    2016-01-01

    Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth. PMID:27214583

  20. Insights into Reference Point Indentation Involving Human Cortical Bone: Sensitivity to Tissue Anisotropy and Mechanical Behavior

    PubMed Central

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

    2014-01-01

    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 10 N 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 (~ 2 mm × 5 mm × 40 mm) 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 (r2=0.25, p=0.003) and toughness (r2=0.35, p=0

  1. Quantification of telomerase activity in normal oral mucosal tissue and oral squamous cell carcinoma

    PubMed Central

    Rai, Arpita; Naikmasur, Venkatesh G.; Sattur, Atul

    2016-01-01

    Background and Objective: The role of telomeres and telomerase in oral cancer is an area of much recent interest. The understanding of the role of telomere biology, the end replication problem leading to genomic instability and the reactivation of telomerase, is absolutely critical to our understanding of oral cancer, and more so, to our ability of early diagnosis and developing novel therapies and cancer prevention approaches. The aim of the present study was to quantify telomerase activity (TA) in oral squamous cell carcinoma (OSCC) and normal oral mucosa and assess the role of telomerase as diagnostic and prognostic marker of oral malignancy. Materials and Methods: We quantified TA in 45 patients with OSCC and 20 normal oral mucosal specimens using polymerase chain reaction-based telomeric repeat amplification protocol assay and compared it with the clinical status and grade of malignancy. Results: TA was detected in 89% of malignant and 5% of normal oral mucosal tissue. The TA levels ranged from 0.28 to 6.91 (mean 2.05, standard deviation [SD] 1.33) in OSCC and 0.21 to 1.09 (mean 0.54, SD 0.27) in normal oral mucosa. There was no relationship between TA levels and clinical stages, site of the lesion, history of adverse habits, or sex of the patient. However, under the WHO classification, there were significant differences (P < 0.00) between Grades I, II, and III. Furthermore, increasing age of the patient significantly correlated with TA. Interpretation and Conclusion: The results of the present study indicate that activation of TA is frequent in OSCC. Statistically significant difference in quantified telomerase levels of OSCC and normal oral mucosa (P < 0.00) demonstrates the significant clinical usefulness of telomerase activation as a valuable marker for diagnosis while significant correlation of TA with grades of malignancy indicates its effectiveness as marker for prognosis of OSCC.

  2. Between-slice intervals in quantification of adipose tissue and muscle in children

    PubMed Central

    Shen, Wei; Chen, Jun; Kwak, Sofia; Punyanitya, Mark; Heymsfield, Steven B.

    2010-01-01

    Magnetic Resonance Imaging (MRI) is increasingly being used in children to quantify adipose tissue (AT) and skeletal muscle (SM) in vivo. it is unclear whether the every 5 cm whole body MRI protocol used in adults is appropriate when applied in children. Whole body MRI continuous 1 cm thick slices were acquired in 73, aged 5–17-year-old healthy children. images were segmented into subcutaneous (SAT), visceral (VAT), intermuscular At (IMAT), and SM. the percentage difference between volumes measured by the continuous protocol and volumes estimated with protocols of different between-slice intervals (i.e., interval = 2, 3, 4 and 5 cm) was larger with an increase in interval size, depot size, weight and body mass index percentile. For group comparisons, studies will require less than 5.4% more subjects if an every 5 cm protocol is used for equivalent power as the every 1 cm protocol. For individual subject comparisons, interval protocols can be used to reliably distinguish between subjects who differ in SM or SAT volume by 0.14 to 0.64 l (i.e., 1 to 5% of SM or SAT volume) or more, or in VAT or IMAT volume by 0.06 to 0.21 l (i.e., 10 to 30% of VAT or IMAT volume) or more. the every 5 cm image acquisition protocol can be considered as accurate as the contiguous protocol for group comparisons in children, as well as for comparison of SM and SAT among individual children. however, a smaller slice interval protocol would be more accurate for comparison of VAT or IMAT among individual children. PMID:20528126

  3. 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

  4. Assessment of a 1H high-resolution magic angle spinning NMR spectroscopy procedure for free sugars quantification in intact plant tissue.

    PubMed

    Delgado-Goñi, Teresa; Campo, Sonia; Martín-Sitjar, Juana; Cabañas, Miquel E; San Segundo, Blanca; Arús, Carles

    2013-08-01

    In most plants, sucrose is the primary product of photosynthesis, the transport form of assimilated carbon, and also one of the main factors determining sweetness in fresh fruits. Traditional methods for sugar quantification (mainly sucrose, glucose and fructose) require obtaining crude plant extracts, which sometimes involve substantial sample manipulation, making the process time-consuming and increasing the risk of sample degradation. Here, we describe and validate a fast method to determine sugar content in intact plant tissue by using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR). The HR-MAS NMR method was used for quantifying sucrose, glucose and fructose in mesocarp tissues from melon fruits (Cucumis melo var. reticulatus and Cucumis melo var. cantalupensis). The resulting sugar content varied among individual melons, ranging from 1.4 to 7.3 g of sucrose, 0.4-2.5 g of glucose; and 0.73-2.83 g of fructose (values per 100 g fw). These values were in agreement with those described in the literature for melon fruit tissue, and no significant differences were found when comparing them with those obtained using the traditional, enzymatic procedure, on melon tissue extracts. The HR-MAS NMR method offers a fast (usually <30 min) and sensitive method for sugar quantification in intact plant tissues, it requires a small amount of tissue (typically 50 mg fw) and avoids the interferences and risks associated with obtaining plant extracts. Furthermore, this method might also allow the quantification of additional metabolites detectable in the plant tissue NMR spectrum.

  5. Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes.

    PubMed

    Zhou, Yi-Hong; Raj, Vinay R; Siegel, Eric; Yu, Liping

    2010-08-16

    In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR) because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR) to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients-a process that requires large sample sizes by combining independent sets of data.

  6. 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.

  7. Quantification of biomineralization: An in-vitro tissue culture system and microanalysis of calcium, phosphorus and trace elements by total-reflection X-ray fluorescence

    SciTech Connect

    Niemann, A.; von Bohlen, A.; Klockenkaemper, R.K.; Keck, E. )

    1990-08-16

    An in-vitro tissue culture system with folded periostea of 17-day-old fetal chick calvaria was combined with analytical methods to achieve quantification of biomineralization. A scanning electron microscope with an energy-dispersive X-ray detector was applied to show the distribution of calcium, phosphorus and trace elements. Calcium and phosphorus were concentrated in the zone of the mineralized matrix. Strontium was distributed similar to calcium. Zinc was distributed equally in the soft tissue and the mineralized matrix. Total-reflection X-ray fluorescence was used for quantification. Thyroxine in high concentration reduces the calcium content of the samples. One week after incubation magnesium chloride (1.8 mM) or zinc chloride (.1 mM) were found to reduce the calcium content by 38% or 82%, respectively.

  8. 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

  9. 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.

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

    PubMed

    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

    2016-09-01

    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 (MR(1)-MR(4)), 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 MR(4.5) 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 MR(4.5) 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

  11. 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.

  12. 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.

  13. 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.

  14. Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue.

    PubMed

    Zhang, Wan-Xia; Fan, Jie; Ma, Jing; Rao, Yi-Song; Zhang, Li; Yan, You-E

    2016-06-22

    Quantitative real-time PCR (qRT-PCR) is the most classical technique in the field of gene expression study. This method requires an appropriate reference gene to normalize mRNA levels. In this study, the expression stability of four frequently-used reference genes in epididymal white adipose tissue (eWAT), inguinal beige adipose tissue (iBeAT) and brown adipose tissue (BAT) from obese and lean rats were evaluated by geNorm, NormFinder and BestKeeper. Based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, the two most stable reference genes were recommended in each type of adipose tissue. Two target genes were applied to test the stability of the reference genes. The geNorm and NormFinder results revealed that GAPDH and 36B4 exhibited the highest expression stabilities in eWAT, while 36B4 and β-actin had the highest expression stabilities in iBeAT and BAT. According to the results of the BestKeeper analysis, 36B4 was the most stable gene in eWAT, iBeAT and BAT, in terms of the coefficient of variance. In terms of the coefficient of correlation, GAPDH, 36B4 and β-actin were the most stable genes in eWAT, iBeAT and BAT, respectively. Additionally, expected results and statistical significance were obtained using a combination of two suitable reference genes for data normalization. In conclusion, 36B4 and GAPDH, in combination, are the best reference genes for eWAT, while 36B4 and β-actin are two most suitable reference genes for both iBeAT and BAT. We recommend using these reference genes accordingly.

  15. Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue

    PubMed Central

    Zhang, Wan-Xia; Fan, Jie; Ma, Jing; Rao, Yi-Song; Zhang, Li; Yan, You-E

    2016-01-01

    Quantitative real-time PCR (qRT-PCR) is the most classical technique in the field of gene expression study. This method requires an appropriate reference gene to normalize mRNA levels. In this study, the expression stability of four frequently-used reference genes in epididymal white adipose tissue (eWAT), inguinal beige adipose tissue (iBeAT) and brown adipose tissue (BAT) from obese and lean rats were evaluated by geNorm, NormFinder and BestKeeper. Based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, the two most stable reference genes were recommended in each type of adipose tissue. Two target genes were applied to test the stability of the reference genes. The geNorm and NormFinder results revealed that GAPDH and 36B4 exhibited the highest expression stabilities in eWAT, while 36B4 and β-actin had the highest expression stabilities in iBeAT and BAT. According to the results of the BestKeeper analysis, 36B4 was the most stable gene in eWAT, iBeAT and BAT, in terms of the coefficient of variance. In terms of the coefficient of correlation, GAPDH, 36B4 and β-actin were the most stable genes in eWAT, iBeAT and BAT, respectively. Additionally, expected results and statistical significance were obtained using a combination of two suitable reference genes for data normalization. In conclusion, 36B4 and GAPDH, in combination, are the best reference genes for eWAT, while 36B4 and β-actin are two most suitable reference genes for both iBeAT and BAT. We recommend using these reference genes accordingly. PMID:27338366

  16. Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue.

    PubMed

    Zhang, Wan-Xia; Fan, Jie; Ma, Jing; Rao, Yi-Song; Zhang, Li; Yan, You-E

    2016-01-01

    Quantitative real-time PCR (qRT-PCR) is the most classical technique in the field of gene expression study. This method requires an appropriate reference gene to normalize mRNA levels. In this study, the expression stability of four frequently-used reference genes in epididymal white adipose tissue (eWAT), inguinal beige adipose tissue (iBeAT) and brown adipose tissue (BAT) from obese and lean rats were evaluated by geNorm, NormFinder and BestKeeper. Based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, the two most stable reference genes were recommended in each type of adipose tissue. Two target genes were applied to test the stability of the reference genes. The geNorm and NormFinder results revealed that GAPDH and 36B4 exhibited the highest expression stabilities in eWAT, while 36B4 and β-actin had the highest expression stabilities in iBeAT and BAT. According to the results of the BestKeeper analysis, 36B4 was the most stable gene in eWAT, iBeAT and BAT, in terms of the coefficient of variance. In terms of the coefficient of correlation, GAPDH, 36B4 and β-actin were the most stable genes in eWAT, iBeAT and BAT, respectively. Additionally, expected results and statistical significance were obtained using a combination of two suitable reference genes for data normalization. In conclusion, 36B4 and GAPDH, in combination, are the best reference genes for eWAT, while 36B4 and β-actin are two most suitable reference genes for both iBeAT and BAT. We recommend using these reference genes accordingly. PMID:27338366

  17. 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.

  18. (18)F Sodium Fluoride PET/CT in Patients with Prostate Cancer: Quantification of Normal Tissues, Benign Degenerative Lesions, and Malignant Lesions.

    PubMed

    Oldan, Jorge D; Hawkins, A Stewart; Chin, Bennett B

    2016-01-01

    Understanding the range and variability of normal, benign degenerative, and malignant (18)F sodium fluoride ((18)F NaF) positron emission tomography/computed tomography (PET/CT) uptake is important in influencing clinical interpretation. Further, it is essential for the development of realistic semiautomated quantification techniques and simulation models. The purpose of this study is to determine the range of these values in a clinically relevant patient population with prostate cancer. (18)F NaF PET/CT scans were analyzed in patients with prostate cancer (n = 47) referred for evaluation of bone metastases. Mean and maximum standardized uptake values [SUVs (SUVmean and SUVmax)] were made in normal background regions (n = 470) including soft tissues (liver, aorta, bladder, adipose, brain, and paraspinal muscle) and osseous structures (T12 vertebral body, femoral diaphyseal cortex, femoral head medullary space, and ribs). Degenerative joint disease (DJD; n = 281) and bone metastases (n = 159) were identified and quantified by an experienced reader using all scan information including coregistered CT. For normal bone regions, the highest (18)F NaF PET SUVmean occurred in T12 (6.8 ± 1.4) and it also showed the lowest coefficient of variation (cv = 21%). For normal soft tissues, paraspinal muscles showed very low SUVmean (0.70 ± 0.11) and also showed the lowest variability (cv = 16%). Average SUVmean in metastatic lesions is higher than uptake in benign degenerative lesions but values showed a wide variance and overlapping values (16.3 ± 13 vs 11.1 ± 3.8; P < 0.00001). The normal (18)F NaF PET uptake values for prostate cancer patients in normal background, benign degenerative disease, and osseous metastases are comparable to those reported for a general population with a wide variety of diagnoses. These normal ranges, specifically for prostate cancer patients, will aid in clinical interpretation and also help to establish the basis of normal limits in a

  19. 18F Sodium Fluoride PET/CT in Patients with Prostate Cancer: Quantification of Normal Tissues, Benign Degenerative Lesions, and Malignant Lesions

    PubMed Central

    Oldan, Jorge D.; Hawkins, A. Stewart; Chin, Bennett B.

    2016-01-01

    Understanding the range and variability of normal, benign degenerative, and malignant 18F sodium fluoride (18F NaF) positron emission tomography/computed tomography (PET/CT) uptake is important in influencing clinical interpretation. Further, it is essential for the development of realistic semiautomated quantification techniques and simulation models. The purpose of this study is to determine the range of these values in a clinically relevant patient population with prostate cancer. 18F NaF PET/CT scans were analyzed in patients with prostate cancer (n = 47) referred for evaluation of bone metastases. Mean and maximum standardized uptake values [SUVs (SUVmean and SUVmax)] were made in normal background regions (n = 470) including soft tissues (liver, aorta, bladder, adipose, brain, and paraspinal muscle) and osseous structures (T12 vertebral body, femoral diaphyseal cortex, femoral head medullary space, and ribs). Degenerative joint disease (DJD; n = 281) and bone metastases (n = 159) were identified and quantified by an experienced reader using all scan information including coregistered CT. For normal bone regions, the highest 18F NaF PET SUVmean occurred in T12 (6.8 ± 1.4) and it also showed the lowest coefficient of variation (cv = 21%). For normal soft tissues, paraspinal muscles showed very low SUVmean (0.70 ± 0.11) and also showed the lowest variability (cv = 16%). Average SUVmean in metastatic lesions is higher than uptake in benign degenerative lesions but values showed a wide variance and overlapping values (16.3 ± 13 vs 11.1 ± 3.8; P < 0.00001). The normal 18F NaF PET uptake values for prostate cancer patients in normal background, benign degenerative disease, and osseous metastases are comparable to those reported for a general population with a wide variety of diagnoses. These normal ranges, specifically for prostate cancer patients, will aid in clinical interpretation and also help to establish the basis of normal limits in a semiautomated data

  20. 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

  1. Reference Genes Selection for Quantitative Real-Time PCR Using RankAggreg Method in Different Tissues of Capra hircus

    PubMed Central

    Najafpanah, Mohammad Javad; Sadeghi, Mostafa; Bakhtiarizadeh, Mohammad Reza

    2013-01-01

    Identification of reference genes with stable levels of gene expression is an important prerequisite for obtaining reliable results in analysis of gene expression data using quantitative real time PCR (RT-qPCR). Since the underlying assumption of reference genes is that expressed at the exact same level in all sample types, in this study, we evaluated the expression stability of nine most commonly used endogenous controls (GAPDH, ACTB, 18S rRNA, RPS18, HSP-90, ALAS, HMBS, ACAC, and B2M) in four different tissues of the domestic goat, Capra hircus, including liver, visceral, subcutaneous fat and longissimus muscles, across different experimental treatments (a standard diet prepared using the NRC computer software as control and the same diet plus one mg chromium/day). We used six different software programs for ranking of reference genes and found that individual rankings of the genes differed among them. Additionally, there was a significant difference in ranking patterns of the studied genes among different tissues. A rank aggregation method was applied to combine the ranking lists of the six programs to a consensus ranking. Our results revealed that HSP-90 was nearly always among the two most stable genes in all studied tissues. Therefore, it is recommended for accurate normalization of RT-qPCR data in goats, while GAPDH, ACTB, and RPS18 showed the most varied expressions and should be avoided as reference genes. PMID:24358246

  2. Development and validation of a specific and sensitive HPLC-ESI-MS method for quantification of lysophosphatidylinositols and evaluation of their levels in mice tissues.

    PubMed

    Masquelier, Julien; Muccioli, Giulio G

    2016-07-15

    Increasing evidence suggests that lysophosphatidylinositols (LPIs), a subspecies of lysophospholipids, are important endogenous mediators. Although LPIs long remained among the less studied lysophospholipids, the identification of GPR55 as their molecular target sparked a renewed interest in the study of these bioactive lipids. Furthermore, increasing evidence points towards a role for LPIs in cancer development. However, a better understanding of the role and functions of LPIs in physiology and disease requires methods that allow for the quantification of LPI levels in cells and tissues. Because dedicated efficient methods for quantifying LPIs were missing, we decided to develop and validate an HPLC-ESI-MS method for the quantification of LPI species from tissues. LPIs are extracted from tissues by liquid/liquid extraction, pre-purified by solid-phase extraction, and finally analyzed by HPLC-ESI-MS. We determined the method's specificity and selectivity, we established calibration curves, determined the carry over (< 2%), LOD and LLOQ (between 0.116-7.82 and 4.62-92.5pmol on column, respectively), linearity (0.988 80%), intermediate precision (CV<20%) as well as the recovery from tissues. We then applied the method to determine the relative abundance of the LPI species in 15 different mouse tissues. Finally, we quantified the absolute LPI levels in six different mouse tissues. We found that while 18:0 LPI represents more than 60% of all the LPI species in the periphery (e.g. liver, gastrointestinal tract, lungs, spleen) it is much less abundant in the central nervous system where the levels of 20:4 LPI are significantly higher. Thus this validated HPLC-ESI-MS method for quantifying LPIs represents a powerful tool that will facilitate the comprehension of the pathophysiological roles of LPIs.

  3. 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). PMID:25036192

  4. 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).

  5. 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.

  6. 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).

  7. 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. PMID:19398033

  8. Quantification of 8-α-hydroxy-mutilin as marker residue for tiamulin in rabbit tissues by high-performance liquid chromatography-mass spectrometry.

    PubMed

    De Baere, Siegrid; Devreese, Mathias; Maes, An; De Backer, Patrick; Croubels, Siska

    2015-06-01

    For the first time, a sensitive and specific method was developed and fully validated for the quantification of the EU marker residue of tiamulin, 8-α-hydroxy-mutilin, in rabbit muscle and liver tissues using liquid chromatography combined with positive heated electrospray ionization triple quadrupole mass spectrometry. The mass spectrometer was operated in the selected reaction monitoring (SRM) mode with selection of the [M + H](+) ion in both quadrupoles 1 and 3, resulting in the SRM transition m/z 337.25 > 337.25 for quantification. Chromatography was performed using a Hypersil Gold C18 column using a gradient elution program with water and methanol as mobile phases. The sample preparation procedure for the analysis of 8-α-hydroxy-mutilin in liver and muscle samples consisted of three main steps: (1) extraction of the tissue matrix using 0.1 N hydrochloric acid/acetone (50/50, v/v), (2) hydrolysis of tiamulin and metabolites to 8-α-hydroxy-mutilin in alkaline medium at 45 °C, and (3) liquid-liquid extraction in acidic medium using ethyl acetate. This is the first method presenting fully validated results, encompassing a linearity of 50 to 2,000 μg/kg, within-run and between-run accuracy and precision, limit of quantification (50 μg/kg for both muscle and liver tissues), limit of detection (muscle, 11.9 μg/kg; liver, 20.6 μg/kg), extraction recovery (muscle, 66.2%; liver, 75.5%), signal suppression and enhancement (muscle, 51.7%; liver, 43.3%), carryover, applicability and practicability, and stability during storage and analysis. This novel method is therefore sensitive enough to be used for residue depletion studies of tiamulin in rabbits and for food safety monitoring with respect to MRL compliance of residues.

  9. Rapid, sensitive and selective HPLC-MS/MS method for the quantification of topically applied besifloxacin in rabbit plasma and ocular tissues: Application to a pharmacokinetic study.

    PubMed

    Gu, Xiao-Fei; Mao, Bai-Yang; Xia, Min; Yang, Yang; Zhang, Jia-Li; Yang, Da-Song; Wu, Wei-Xin; Du, Ying-Xiang; Di, Bin; Su, Meng-Xiang

    2016-01-01

    Besifloxacin is a fourth-generation broad-spectrum fluoroquinolone registered for the topical treatment of bacterial conjunctivitis. In this study, a rapid, sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for quantification of besifloxacin in rabbit plasma and ocular tissues using nateglinide as the internal standard (IS). The analyte and IS were separated on a Sepax GP-Phenyl column by isocratic elution with methanol-acetonitrile-5 mM ammonium formate-formic acid (29:55:16:0.1, v/v/v/v) as the mobile phase at a flow rate of 1.2 mL/min, and the total run time was 3.0 min. An electrospray ionization (ESI) source was applied and operated in the positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification, and the monitored transitions were 394.2→377.1 for besifloxacin and m/z 318.3→166.1 for the IS. The calibration curve was linear over the range of 0.103-206 ng/mL for plasma and 2.06-2060 ng/mL for tears, aqueous humor, conjunctiva and cornea with correlation coefficient (r) greater than 0.99. The lower limit of quantification (LLOQ) for besifloxacin was 0.103 ng/mL for plasma and 2.06 ng/mL for other ocular tissues with good accuracy and precision. Intra- and inter-batch precision were both lower than 15% and accuracy ranged from 85% to 115% at all QC levels. The method was successfully applied to the pharmacokinetic study of besifloxacin in rabbit plasma and ocular tissues after single and multiple topical administrations. PMID:26340560

  10. 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

  11. 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.

  12. Feasibility studies into the production of gamma-irradiated oyster tissue reference materials for paralytic shellfish poisoning toxins.

    PubMed

    Turner, Andrew D; Lewis, Adam M; Hatfield, Robert G; Powell, Andy L; Higman, Wendy A

    2013-09-01

    A study was conducted to assess the feasibility for the production of sterile, stable and homogenous shellfish reference materials containing known concentrations of paralytic shellfish poisoning (PSP) toxins. Pacific oysters were contaminated with toxins following mass culturing of toxic algae and shellfish feeding experiments. Live oysters were shucked and tissues homogenised, before measuring into multiple aliquots, with one batch subjected to gamma irradiation treatment and the other remaining untreated. The homogeneity of both batches of samples was assessed using a pre-column oxidation liquid chromatography with fluorescence detection (Pre-COX LC-FLD) method and shown to be within the limits of normal within-batch repeatability. A twelve-month stability experiment was conducted for both untreated and gamma irradiated batches, specifically examining the effects of long term storage at -20 °C, +4 °C and +40 °C. Results indicated mostly good stability of PSP toxins in both materials when stored frozen at -20 °C, but with the instability of GTX2&3 concentrations in the untreated tissues eliminated in the irradiated tissues. Analysis using a post-column oxidation (PCOX) LC-FLD method also showed epimerisation in both GTX1&4 and GTX2&3 epimeric pairs in untreated samples after only 6 months frozen storage. This issue was not present in the tissues irradiated before long term storage. Biological activity testing confirmed the absence of bacteria in the irradiated samples throughout the 12 month study period. With such results there was clear evidence for the potential of increasing the scale of the mass culturing and shellfish feeding for the production of large batches of tissue suitable for the preparation of a certified matrix reference material. Overall results demonstrated the feasibility for production of oyster reference materials for PSTs, with evidence for prolonged stability following gamma irradiation treatment and storage at -20 °C.

  13. Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta

    PubMed Central

    Rueda-Martínez, Carmen; Lamas, Oscar; Mataró, María José; Robledo-Carmona, Juan; Sánchez-Espín, Gemma; Jiménez-Navarro, Manuel; Such-Martínez, Miguel; Fernández, Borja

    2014-01-01

    Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples. PMID:24841551

  14. Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits

    PubMed Central

    2011-01-01

    Background Botrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for quantification of B. cinerea in apple (Red Delicious), table grape (pink Moscatel), and pear (William's) tissues. Results The method was based in the competition for the binding site of monoclonal antibodies between B. cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1 B. cinerea antigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation. Conclusions The developed method allowed detects the presence of B. cinerea in asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide. PMID:21970317

  15. Mitoxantrone loaded superparamagnetic nanoparticles for drug targeting: a versatile and sensitive method for quantification of drug enrichment in rabbit tissues using HPLC-UV.

    PubMed

    Tietze, Rainer; Schreiber, Eveline; Lyer, Stefan; Alexiou, Christoph

    2010-01-01

    In medicine, superparamagnetic nanoparticles bound to chemotherapeutics are currently investigated for their feasibility in local tumor therapy. After intraarterial application, these particles can be accumulated in the targeted area by an external magnetic field to increase the drug concentration in the region of interest (Magnetic-Drug-Targeting). We here present an analytical method (HPLC-UV), to detect pure or ferrofluid-bound mitoxantrone in a complex matrix even in trace amounts in order to perform biodistribution studies. Mitoxantrone could be extracted in high yields from different tissues. Recovery of mitoxantrone in liver tissue (5000 ng/g) was 76 +/- 2%. The limit of quantification of mitoxantrone standard was 10 ng/mL +/-12%. Validation criteria such as linearity, precision, and stability were evaluated in ranges achieving the FDA requirements. As shown for pilot samples, biodistribution studies can easily be performed after application of pure or ferrofluid-bound mitoxantrone. PMID:20490266

  16. 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. PMID:20016988

  17. Comparison of quantification methods to measure fire-derived (black/elemental) carbon in soils and sediments using reference materials from soil, water, sediment and the atmosphere

    NASA Astrophysics Data System (ADS)

    Hammes, Karen; Schmidt, Michael W. I.; Smernik, Ronald J.; Currie, Lloyd A.; Ball, William P.; Nguyen, Thanh H.; Louchouarn, Patrick; Houel, Stephane; Gustafsson, Örjan; Elmquist, Marie; Cornelissen, Gerard; Skjemstad, Jan O.; Masiello, Caroline A.; Song, Jianzhong; Peng, Ping'an; Mitra, Siddhartha; Dunn, Joshua C.; Hatcher, Patrick G.; Hockaday, William C.; Smith, Dwight M.; Hartkopf-Fröder, Christoph; BöHmer, Axel; Lüer, Burkhard; Huebert, Barry J.; Amelung, Wulf; Brodowski, Sonja; Huang, Lin; Zhang, Wendy; Gschwend, Philip M.; Flores-Cervantes, D. Xanat; Largeau, Claude; Rouzaud, Jean-NoëL.; Rumpel, Cornelia; Guggenberger, Georg; Kaiser, Klaus; Rodionov, Andrei; Gonzalez-Vila, Francisco J.; Gonzalez-Perez, José A.; de La Rosa, José M.; Manning, David A. C.; López-CapéL, Elisa; Ding, Luyi

    2007-09-01

    to provide a basis on which to choose between the different BC quantification methods in soil and sediment studies. In this paper we summarize the advantages and disadvantages of each method. In future studies, we strongly recommend the evaluation of all methods analyzing for BC in soils and sediments against the set of BC reference materials analyzed here.

  18. 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.

  19. Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry

    PubMed Central

    Seibert, Cathrin; Davidson, Brian R.; Fuller, Barry J.; Patterson, Laurence H.; Griffiths, William J.; Wang, Yuqin

    2009-01-01

    Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labelled tryptic peptide and analysed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labelled tryptic peptides and their natural unlabelled analogues quantification could be performed over the range of 0.1 – 1.5 pmol on column. Liver microsomes from four individuals were analysed for CYP2E1 giving values of 88 - 200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 – 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP-isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP-isoforms in a single sample. PMID:19714871

  20. 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. PMID:16846144

  1. 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

  2. Quantification of branched-chain keto acids in tissue by ultra fast liquid chromatography-mass spectrometry.

    PubMed

    Olson, Kristine C; Chen, Gang; Lynch, Christopher J

    2013-08-15

    Branched-chain keto acids (BCKAs) are associated with increased susceptibility to several degenerative diseases. However, BCKA concentrations in tissues or the amounts of tissue available are frequently at the limit of detection for standard plasma methods. To accurately and quickly determine tissue BCKAs, we have developed a sensitive ultra fast liquid chromatography-mass spectrometry (UFLC-MS) method. BCKAs from deproteinized tissue extractions were o-phenylenediamine (OPD) derivatized, ethyl acetate extracted, lyophilized in a vacuum centrifuge, and reconstituted in 200 mM ammonium acetate. Samples were injected onto a Shimadzu UFLC system coupled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument that detected masses of the OPD BCKA products using a multiple reaction monitoring method. An OPD-derivatized (13)C-labeled keto acid was used as an internal standard. Application of the method for C57BL/6J (wild-type) and PP2Cm knockout mouse tissues, including kidney, adipose tissue, liver, gastrocnemius, and hypothalamus, is shown. The lowest tissue concentration measured by this method was 20 nM, with the standard curve covering a wide range (7.8-32,000 nM). Liquid chromatography-mass spectrometry run times for this assay were less than 5 min, facilitating high throughput, and the OPD derivatives were found to be stable over several days.

  3. Separation and quantification of monothiols and phytochelatins from a wide variety of cell cultures and tissues of trees and other plants using high performance liquid chromatography.

    PubMed

    Minocha, Rakesh; Thangavel, P; Dhankher, Om Parkash; Long, Stephanie

    2008-10-17

    The HPLC method presented here for the quantification of metal-binding thiols is considerably shorter than most previously published methods. It is a sensitive and highly reproducible method that separates monobromobimane tagged monothiols (cysteine, glutathione, gamma-glutamylcysteine) along with polythiols (PC(2), PC(3), PC(4) and PC(5)) within 23min from a wide variety of samples. Total run time of the method is 35min. Detection limits for thiols is 33fmol for 10microlL injection. This method will be applicable to study the metal detoxification mechanisms for a wide variety of cell cultures and tissues of plants and trees including algae, Arabidopsis, crambe, rice, and red spruce. PMID:18760414

  4. Separation and quantification of monothiols and phytochelatins from a wide variety of cell cultures and tissues of trees and other plants using high performance liquid chromatography.

    PubMed

    Minocha, Rakesh; Thangavel, P; Dhankher, Om Parkash; Long, Stephanie

    2008-10-17

    The HPLC method presented here for the quantification of metal-binding thiols is considerably shorter than most previously published methods. It is a sensitive and highly reproducible method that separates monobromobimane tagged monothiols (cysteine, glutathione, gamma-glutamylcysteine) along with polythiols (PC(2), PC(3), PC(4) and PC(5)) within 23min from a wide variety of samples. Total run time of the method is 35min. Detection limits for thiols is 33fmol for 10microlL injection. This method will be applicable to study the metal detoxification mechanisms for a wide variety of cell cultures and tissues of plants and trees including algae, Arabidopsis, crambe, rice, and red spruce.

  5. [THE MARKERS OF BONE TISSUE METABOLISM. THE REFERENCE VALUES FOR THE KHANTY-MANSI AUTONOMOUS OKRUG-YUGRA].

    PubMed

    Kutchin, R V; Nenenko, N D; Tchernitsina, N V; Maksimova, T A

    2016-03-01

    The article defines reference values of particular markers of metabolism of bone tissue common to residents of the Khanty-Mansi Autonomous Okrug-Yugra. The enzyme-linked immunosorbent assay was applied to analyze blood serum of 86 patients (43 males, 43 females) detecting concentration of C-tailed telopeptide of collagen type I, osteocalcin, calcitonin, parathyroid hormone and 1.25(OH)2 vitamin D. The following reference values were derived. The C-tailed telopeptide (ng/ml): 0.111 (0.071-0.162) for females and 0.146 (0.066-0.255) for males. The osteocalcin (ng/ml): 20.6 (12.9-33.0) for females and 27.6 (12.0-61.9) for males. Calcitonin (pg/ml) - 2.55 (1.90-3.76); parathyroid hormone (pg/ml) - 39 (13-88); 1.25(OH)2 vitamin D (pg/ml) - 10.5 (3.9-46.4). It was also noted that decreasing of average indicators of vitamin D level and increasing of level of parathyroid hormone among residents of the Khanty-Mansi Autonomous Okrug-Yugra can cause increasing of intensity of accumulation of minerals in bone tissue as compared with residents of middle latitudes. PMID:27506104

  6. Comparative tissue distribution of heavy metals in house sparrow (Passer domesticus, Aves) in polluted and reference sites in Turkey.

    PubMed

    Albayrak, Tamer; Mor, Firdevs

    2011-10-01

    Bioindicators are useful for environmental monitoring in ecosystems with pollution loads. We compared concentrations of selected 10 metals in 42 samples of House Sparrow in a polluted by thermal power plant and reference sites. We found mean tissue concentrations of some metals to be significantly higher in sparrows from the polluted area when compared to tissues from the reference site. In liver mean concentrations of Cu (35.85 ± 17.22 mg kg(-1)) and Zn (101.76 ± 26.38 mg kg(-1)) were significantly higher and concentration of Ni (0.43 ± 0.49 mg kg(-1)) were significantly lower in sparrows from the polluted area (p<0.05). The concentration of Cu was significantly higher in muscle and liver at the polluted site. Gender did not seem to influence residue levels, of the elements studied, among sparrows with the exception of kidney cobalt concentrations; which were higher in female sparrows than in males (p<0.05, t=-2.409).

  7. Development of an immunohistochemical protein quantification system in conjunction with tissue microarray technology for identifying predictive biomarkers for phosphatidylinositol 3-kinase inhibitors.

    PubMed

    Isoyama, Sho; Yoshimi, Hisashi; Dan, Shingo; Okamura, Mutsumi; Seki, Mariko; Irimura, Tatsuro; Yamori, Takao

    2012-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers by gain-of-function mutations of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) or dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Therefore PI3K is thought to be a promising target for cancer therapy. Many agents targeting PI3K have been developed and some of them have been evaluated in clinical trials. In recent years, development of predictive biomarkers as companion diagnostics for molecular targeted drugs has become an important requirement for clinical development; however, no clinically established biomarkers that predict the efficacy of PI3K inhibitors have been found. We previously reported that expression of phosphorylated Akt determined by immunoblot analysis correlated with the antitumor efficacy of a PI3K inhibitor ZSTK474 in vitro and in vivo, suggesting that it might be used as a predictive biomarker. In this study, to evaluate biomarker candidates in in vivo tumor samples, we developed an immunohistochemical protein detection/quantification system in conjunction with the tissue microarray technology using a panel of 24 human tumor xenografts (JFCR24). We have clearly demonstrated that expression levels of phosphorylated v-akt murine thymoma viral oncogene homolog (Akt) and mitogen-activated protein kinase (MAPK) determined by this system significantly correlated with those determined by immunoblot analysis. As expected, PTEN status correlated with expression of phosphorylated Akt but not MAPK. Finally, we confirmed that phosphorylated Akt levels determined using this system correlated with the in vivo efficacy of ZSTK474. The present results indicate that the immunohistochemical protein detection/quantification system could be used to quantify expression of biomarker proteins in xenografted tumor tissues as well as in human tumor specimens to predict drug efficacy in future clinical trials. PMID:22975517

  8. 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.

  9. Chemistry, nutritional sources, tissue distribution and metabolism of vitamin K with special reference to bone health.

    PubMed

    Shearer, M J; Bach, A; Kohlmeier, M

    1996-04-01

    Vitamin K occurs in nature as a series of compounds with a common 2-methyl- 1,4 naphthoquinone nucleus and differing isoprenoid side chains at the 3 position. They comprise a single major plant form, phylloquinone with a phytyl side chain and a family of bacterially synthesized menaquinones (MKs) with multiprenyl side chains. The major dietary source to humans is phylloquinone for which the chief food contributors are green, leafy vegetables followed by certain vegetable oils (soybean, rapeseed and olive oils). Recent analyses by high pressure liquid chromatography are now providing a wide-ranging database of phylloquinone in foods. Menaquinones are found in moderate concentrations in only a few foods such as cheeses (MK-8 and MK-9). A wider spectrum of MKs is synthesized by the gut microflora, and their intestinal absorption probably accounts for most of the hepatic stores, particularly those with very long side chains (MKs-10--13) synthesized by members of the genus Bacteroides. The site of absorption of floral MKs is not known, but reasonable concentrations are found in the terminal ileum where bile salt-mediated absorption is possible. Both phylloquinone and menaquinones are bioactive in hepatic gamma-carboxylation but long-chain MKs are less well absorbed. Liver stores of vitamin K are relatively small and predominantly MKs-7--13. The hepatic reserves of phylloquinone (approximately 10% of the total) are labile and turn over at a faster rate than menaquinones. Trabecular and cortical bone appear to contain substantial concentrations of both phylloquinone and menaquinones. A majority (approximately 60-70%) of the daily dietary intake of phylloquinone is lost to the body by excretion, which emphasizes the need for a continuous dietary supply to maintain tissue reserves.

  10. 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.

  11. 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.

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

    PubMed

    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 (PrP(CWD)) 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 PrP(CWD) 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 PrP(RES) in prion, and potentially other, protein misfolding disease states. PMID:27157060

  13. 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

  14. Quantification of biological tissue and construction of patient equivalent phantom (skull and chest) for infants (1-5 years old)

    NASA Astrophysics Data System (ADS)

    Alves, A. F.; Pina, D. R.; Bacchim Neto, F. A.; Ribeiro, S. M.; Miranda, J. R. A.

    2014-03-01

    Our main purpose in this study was to quantify biological tissue in computed tomography (CT) examinations with the aim of developing a skull and a chest patient equivalent phantom (PEP), both specific to infants, aged between 1 and 5 years old. This type of phantom is widely used in the development of optimization procedures for radiographic techniques, especially in computed radiography (CR) systems. In order to classify and quantify the biological tissue, we used a computational algorithm developed in Matlab ®. The algorithm performed a histogram of each CT slice followed by a Gaussian fitting of each tissue type. The algorithm determined the mean thickness for the biological tissues (bone, soft, fat, and lung) and also converted them into the corresponding thicknesses of the simulator material (aluminum, PMMA, and air). We retrospectively analyzed 148 CT examinations of infant patients, 56 for skull exams and 92 were for chest. The results provided sufficient data to construct a phantom to simulate the infant chest and skull in the posterior-anterior or anterior-posterior (PA/AP) view. Both patient equivalent phantoms developed in this study can be used to assess physical variables such as noise power spectrum (NPS) and signal to noise ratio (SNR) or perform dosimetric control specific to pediatric protocols.

  15. Validation of an extraction and gas chromatography-mass spectrometry quantification method for cocaine, methadone, and morphine in postmortem adipose tissue.

    PubMed

    Colucci, A P; Aventaggiato, L; Centrone, M; Gagliano-Candela, R

    2010-01-01

    Adipose tissue is a complex biological matrix that necessitates several pre-analytical preparation steps to separate drugs and metabolites from the lipophilic matrix. A novel, sensitive, and specific gas chromatographic-mass spectrometric (GC-MS) method for the determination of cocaine (metabolites), methadone, and morphine in postmortem adipose tissue was developed, optimized, and validated. The method involves the aqueous acid extraction of analytes, alkalinization of the extract, solid-phase extraction with chloroform, and derivatization with BSTFA before GC-MS analysis. Deuterated compounds were used as internal standards for determination and quantification of analytes. Limits of detection were 0.005 microg/g for cocaine and cocaethylene, 0.02 microg/g for benzoylecgonine, 0.01 microg/g for ecgoninemethylester, 0.005 microg/g for methadone, and 0.01 microg/g for morphine. Linearity ranged from 0.1 to 1.000 microg/g for all analytes. Intra- and interday accuracy ranged from 70.6 to 105%, and intra- and interday precisions were less than 8.2% and 8.6%, respectively, for all analytes. The method showed a good recovery. PMID:20663287

  16. An Improved Simplified High-Sensitivity Quantification Method for Determining Brassinosteroids in Different Tissues of Rice and Arabidopsis1[C][W

    PubMed Central

    Xin, Peiyong; Yan, Jijun; Fan, Jinshi; Chu, Jinfang; Yan, Cunyu

    2013-01-01

    Quantification of brassinosteroids is essential and extremely important to study the molecular mechanisms of their physiological roles in plant growth and development. Herein, we present a simple, material and cost-saving high-performance method for determining endogenous brassinosteroids (BRs) in model plants. This new method enables simultaneous enrichment of a wide range of bioactive BRs such as brassinolide, castasterone, teasterone, and typhasterol with ion exchange solid-phase extraction and high-sensitivity quantitation of these BRs based on isotope dilution combined with internal standard approach. For routine analysis, the consumption of plant materials was reduced to one-twentieth of previously reported and the overall process could be completed within 1 day compared with previous 3 to 4 days. The strategy was validated by profiling BRs in different ecotypes and mutants of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the BR distributions in different model plants tissues were determined with the new method. The method allows plant physiologists to monitor the dynamics and distributions of BRs with 1 gram fresh weight of model plant tissues, which will speed up the process for the molecular mechanism research of BRs with these model plants in future work. PMID:23800992

  17. 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

  18. Quantification of age-related changes of α-tocopherol in lysosomal membranes in murine tissues and human fibroblasts.

    PubMed

    König, Jeannette; Besoke, Fabian; Stuetz, Wolfgang; Malarski, Angelika; Jahreis, Gerhard; Grune, Tilman; Höhn, Annika

    2016-05-01

    Considering the biological function of α-tocopherol (α-Toc) as a potent protective factor against oxidative stress, this antioxidant is in the focus of aging research. To understand the role of α-Toc during aging we investigated α-Toc concentrations in young and aged primary human fibroblasts after supplementation with RRR-α-Toc. Additionally, α-Toc contents were determined in brain, kidney, and liver tissue of 10 week-, 18 month-, and 24 month-old mice, which were fed a standard diet containing 100 mg/kg dl-α-tocopheryl acetate. α-Toc concentrations in isolated lysosomes and the expression of the α-Toc transport proteins Niemann Pick C1 (NPC1), Niemann Pick C2 (NPC2), and lipoprotein lipase were also analyzed. Obtained data show a significant age-related increase of α-Toc in murine liver, kidney, and brain tissue as well as in human dermal fibroblasts. Also liver and kidney lysosomes are marked by elevated α-Toc contents with aging. NPC1 and NPC2 protein amounts are significantly decreased in adult and aged murine kidney tissue. Also aged human dermal fibroblasts show decreased NPC1 amounts. Supplementation of young and aged fibroblasts led also to decreased NPC1 amounts, suggesting a direct role of this protein in α-Toc distribution. Our results indicate an age-dependent increase of α-Toc in different murine tissues as well as in human fibroblasts. Furthermore saturation and intracellular distribution of α-Toc seem to be strongly dependent on the availability of this vitamin as well as on the presence of the lysosomal protein NPC1. © 2016 BioFactors, 42(3):307-315, 2016.

  19. 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.

  20. Comparison of Real-Time PCR Signal-Amplified In Situ Hybridization and Conventional PCR for Detection and Quantification of Human Papillomavirus in Archival Cervical Cancer Tissue

    PubMed Central

    Biedermann, Karin; Dandachi, Nadia; Trattner, Maria; Vogl, Georgia; Doppelmayr, Hildegard; Moré, Elena; Staudach, Alfons; Dietze, Otto; Hauser-Kronberger, Cornelia

    2004-01-01

    Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P = 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 (κ = 0.661) and HPV18 (κ = 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification. PMID:15297527

  1. Tryptic peptide reference data sets for MALDI imaging mass spectrometry on formalin-fixed ovarian cancer tissues.

    PubMed

    Meding, Stephan; Martin, Karina; Gustafsson, Ove J R; Eddes, James S; Hack, Sandra; Oehler, Martin K; Hoffmann, Peter

    2013-01-01

    MALDI imaging mass spectrometry is a powerful tool for morphology-based proteomic tissue analysis. However, peptide identification is still a major challenge due to low S/N ratios, low mass accuracy and difficulties in correlating observed m/z species with peptide identities. To address this, we have analyzed tryptic digests of formalin-fixed paraffin-embedded tissue microarray cores, from 31 ovarian cancer patients, by LC-MS/MS. The sample preparation closely resembled the MALDI imaging workflow in order to create representative reference data sets containing peptides also observable in MALDI imaging experiments. This resulted in 3844 distinct peptide sequences, at a false discovery rate of 1%, for the entire cohort and an average of 982 distinct peptide sequences per sample. From this, a total of 840 proteins and, on average, 297 proteins per sample could be inferred. To support the efforts of the Chromosome-centric Human Proteome Project Consortium, we have annotated these proteins with their respective chromosome location. In the presented work, the benefit of using a large cohort of data sets was exemplified by correct identification of several m/z species observed in a MALDI imaging experiment. The tryptic peptide data sets generated will facilitate peptide identification in future MALDI imaging studies on ovarian cancer.

  2. Multiclass Carcinogenic DNA Adduct Quantification in Formalin-Fixed Paraffin-Embedded Tissues by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Guo, Jingshu; Yun, Byeong Hwa; Upadhyaya, Pramod; Yao, Lihua; Krishnamachari, Sesha; Rosenquist, Thomas A; Grollman, Arthur P; Turesky, Robert J

    2016-05-01

    DNA adducts are a measure of internal exposure to genotoxicants and an important biomarker for human risk assessment. However, the employment of DNA adducts as biomarkers in human studies is often restricted because fresh-frozen tissues are not available. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis are readily accessible. Recently, our laboratory reported that DNA adducts of aristolochic acid, a carcinogenic component of Aristolochia herbs used in traditional Chinese medicines worldwide, can be recovered quantitatively from FFPE tissues. In this study, we have evaluated the efficacy of our method for retrieval of DNA adducts from archived tissue by measuring DNA adducts derived from four other classes of human carcinogens: polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic aromatic amines (HAAs), and N-nitroso compounds (NOCs). Deoxyguanosine (dG) adducts of the PAH benzo[a]pyrene (B[a]P), 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N(2)-B[a]PDE); the aromatic amine 4-aminobiphenyl (4-ABP), N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP); the HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP); and the dG adducts of the NOC 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), O(6)-methyl-dG (O(6)-Me-dG) and O(6)-pyridyloxobutyl-dG (O(6)-POB-dG), formed in liver, lung, bladder, pancreas, or colon were recovered in comparable yields from fresh-frozen and FFPE preserved tissues of rodents treated with the procarcinogens. Quantification was achieved by ultraperformance liquid chromatography coupled with electrospray ionization ion-trap multistage mass spectrometry (UPLC/ESI-IT-MS(3)). These advancements in the technology of DNA adduct retrieval from FFPE tissue clear the way for use of archived pathology samples in molecular epidemiology studies designed to assess the causal role of exposure to hazardous chemicals

  3. Influence of Skin and Subcutaneous Tissue on High-Intensity Focused Ultrasound Beam: Experimental Quantification and Numerical Modeling.

    PubMed

    Grisey, Anthony; Heidmann, Marc; Letort, Veronique; Lafitte, Pauline; Yon, Sylvain

    2016-10-01

    High-intensity focused ultrasound (HIFU) enables the non-invasive thermal ablation of tumors. However, numerical simulations of the treatment remain complex and difficult to validate in clinically relevant situations. In this context, needle hydrophone measurements of the acoustic field downstream of seven rabbit tissue layers comprising skin, subcutaneous fat and muscle were performed in different geometrical configurations. Increasing curvature and thickness of the sample were found to decrease the focusing of the beam: typically, a curvature of 0.05 mm(-1) decreased the maximum pressure by 45% and doubled the focal area. A numerical model based on k-Wave Toolbox was found to be in very good agreement with the reported measurements. It was used to extrapolate the effect of the superficial tissues on peak positive and peak negative pressure at focus, which affects both cavitation and target heating. The shape of the interface was found to have a strong influence on the values, and it is therefore an important parameter to monitor or to control in the clinical practice. This also highlights the importance of modeling realistic configurations when designing treatment procedures. PMID:27471120

  4. A strategy for designing multi-taxa specific reference gene systems. example of application--ppi phosphofructokinase (ppi-PPF) used for the detection and quantification of three taxa: maize (Zea mays), cotton (Gossypium hirsutum) and rice (Oryza sativa).

    PubMed

    Chaouachi, Maher; Giancola, Sandra; Romaniuk, Marcel; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2007-10-01

    In the first part of the paper, we report the description of a new strategy for the development of a plant reference gene system that can be used for genetically modified organism (GMO) analysis. On the basis of in silico research for candidate genes, the design of degenerate primers allowed the obtention of genomic sequences of the selected gene ppi-phosphofructokinase ( ppi-PPF) for nine taxa in which GMOs have been developed. The comparison and the analysis of inter- and intraspecies sequence variability were performed using a large number of species and cultivars. As an example of application following the detection of single nucleotide polymorphism, we designed specific conventional and real-time polymerase chain reaction tests for the detection and quantification of three taxa, namely, maize, cotton, and rice. This system was highly specific and sensitive. The gene copy number conservation among different cultivars was analyzed and confirmed with a sequencing step. This reference gene system is adequate for use in routine assays for the quantification of GMOs. We then explain briefly the constraints faced and propose recommendations when designing a reference gene system depending on the species to be targeted. PMID:17824661

  5. A strategy for designing multi-taxa specific reference gene systems. example of application--ppi phosphofructokinase (ppi-PPF) used for the detection and quantification of three taxa: maize (Zea mays), cotton (Gossypium hirsutum) and rice (Oryza sativa).

    PubMed

    Chaouachi, Maher; Giancola, Sandra; Romaniuk, Marcel; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2007-10-01

    In the first part of the paper, we report the description of a new strategy for the development of a plant reference gene system that can be used for genetically modified organism (GMO) analysis. On the basis of in silico research for candidate genes, the design of degenerate primers allowed the obtention of genomic sequences of the selected gene ppi-phosphofructokinase ( ppi-PPF) for nine taxa in which GMOs have been developed. The comparison and the analysis of inter- and intraspecies sequence variability were performed using a large number of species and cultivars. As an example of application following the detection of single nucleotide polymorphism, we designed specific conventional and real-time polymerase chain reaction tests for the detection and quantification of three taxa, namely, maize, cotton, and rice. This system was highly specific and sensitive. The gene copy number conservation among different cultivars was analyzed and confirmed with a sequencing step. This reference gene system is adequate for use in routine assays for the quantification of GMOs. We then explain briefly the constraints faced and propose recommendations when designing a reference gene system depending on the species to be targeted.

  6. A one-vial method for routine extraction and quantification of free fatty acids in blood and tissue by HPLC.

    PubMed

    Jüngling, E; Kammermeier, H

    1988-05-15

    A one-vial procedure has been developed to quantify free fatty acids in human blood serum and rat heart tissue. To allow routine analysis the system has been constructed to allow simultaneous processing of nine samples (Praechromat). The free fatty acids are extracted with Freon 11 and then derivatized to coumarin esters prior to HPLC. The Freon 11 extraction of the free fatty acids is rapid and complete. Neither hydrolytic degradation of natural fatty acid esters nor oxidative damage of unsaturated fatty acids was observed. Fifteen free fatty acids (FFA) were routinely quantified by isocratic elution with high reproducibility (SD less than 4%) and good recovery (0.1 mM FFA: 98-100%, 0.02 mM: 91-105%). The free fatty acids could be determined in the range from 20 pmol to 20 nmol. PMID:3407911

  7. Identification and quantification of bleomycin in serum and tumor tissue by liquid chromatography coupled to high resolution mass spectrometry.

    PubMed

    Kosjek, Tina; Krajnc, Anja; Gornik, Tjasa; Zigon, Dusan; Groselj, Ales; Sersa, Gregor; Cemazar, Maja

    2016-11-01

    Bleomycin is a cytotoxic antibiotic available as a compost of structurally strongly related glycopeptides, which is in vivo found chelated with several metals. Its pharmacotherapy has merely been based on experimental dose - response data, whereas its biodistribution and pharmacokinetics remain fundamentally unknown. This is reasoned by an absence of a specific and sensitive mass spectrometry-based analytical method for its determination in biological tissues. We herein reveal the results of our study on the mass spectrometric behavior of two main bleomycin fractions A2 and B2, including their metal complexes, particularly the predominant copper chelates. In the electrospray ion source bleomycin forms double charged species, where for the metal-free fraction A2 and its copper complex m/z 707.76 and m/z 707.21 are seen, respectively. Hence, the second isotopic ion of the chelate (m/z 707.71) nearly coincides with the first isotopic ion of the metal-free fraction. This phenomenon can only be followed by high-resolution mass spectrometry, and is considered the plausible reason, why the attempts to determine bleomycin with mass spectrometry have been so scarce. The presented paper further describes a sensitive and selective liquid chromatography - mass spectrometry analytical method for determination of bleomycin in serum and tumor tissues. This newly developed method was employed for bleomycin pharmacokinetic studies in serum and tumors of laboratory animals. Additionally, the method was employed for determination of bleomycin pharmacokinetic parameters in elderly patients in order to determine the effective therapeutic window of electrochemotherapy with bleomycin. PMID:27591601

  8. Noninvasive quantification of in vitro osteoblastic differentiation in 3D engineered tissue constructs using spectral ultrasound imaging.

    PubMed

    Gudur, Madhu Sudhan Reddy; Rao, Rameshwar R; Peterson, Alexis W; Caldwell, David J; Stegemann, Jan P; Deng, Cheri X

    2014-01-01

    Non-destructive monitoring of engineered tissues is needed for translation of these products from the lab to the clinic. In this study, non-invasive, high resolution spectral ultrasound imaging (SUSI) was used to monitor the differentiation of MC3T3 pre-osteoblasts seeded within collagen hydrogels. SUSI was used to measure the diameter, concentration and acoustic attenuation of scatterers within such constructs cultured in either control or osteogenic medium over 21 days. Conventional biochemical assays were used on parallel samples to determine DNA content and calcium deposition. Construct volume and morphology were accurately imaged using ultrasound. Cell diameter was estimated to be approximately 12.5-15.5 µm using SUSI, which corresponded well to measurements of fluorescently stained cells. The total number of cells per construct assessed by quantitation of DNA content decreased from 5.6±2.4×10(4) at day 1 to 0.9±0.2×10(4) at day 21. SUSI estimation of the equivalent number of acoustic scatters showed a similar decreasing trend, except at day 21 in the osteogenic samples, which showed a marked increase in both scatterer number and acoustic impedance, suggestive of mineral deposition by the differentiating MC3T3 cells. Estimation of calcium content by SUSI was 41.7±11.4 µg/ml, which agreed well with the biochemical measurement of 38.7±16.7 µg/ml. Color coded maps of parameter values were overlaid on B-mode images to show spatiotemporal changes in cell diameter and calcium deposition. This study demonstrates the use of non-destructive ultrasound imaging to provide quantitative information on the number and differentiated state of cells embedded within 3D engineered constructs, and therefore presents a valuable tool for longitudinal monitoring of engineered tissue development.

  9. 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

  10. Quantification of Endogenous Retinoids

    PubMed Central

    Kane, Maureen A.; Napoli, Joseph L.

    2014-01-01

    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. PMID:20552420

  11. Quantification of longitudinal tissue pO2 gradients in window chamber tumours: impact on tumour hypoxia

    PubMed Central

    Dewhirst, M W; Ong, E T; Braun, R D; Smith, B; Klitzman, B; Evans, S M; Wilson, D

    1999-01-01

    We previously reported that the arteriolar input in window chamber tumours is limited in number and is constrained to enter the tumour from one surface, and that the pO2 of tumour arterioles is lower than in comparable arterioles of normal tissues. On average, the vascular pO2 in vessels of the upper surface of these tumours is lower than the pO2 of vessels on the fascial side, suggesting that there may be steep vascular longitudinal gradients (defined as the decline in vascular pO2 along the afferent path of blood flow) that contribute to vascular hypoxia on the upper surface of the tumours. However, we have not previously measured tissue pO2 on both surfaces of these chambers in the same tumour. In this report, we investigated the hypothesis that the anatomical constraint of arteriolar supply from one side of the tumour results in longitudinal gradients in pO2 sufficient in magnitude to create vascular hypoxia in tumours grown in dorsal flap window chambers. Fischer-344 rats had dorsal flap window chambers implanted in the skin fold with simultaneous transplantation of the R3230AC tumour. Tumours were studied at 9–11 days after transplantation, at a diameter of 3–4 mm; the tissue thickness was 200 μm. For magnetic resonance microscopic imaging, gadolinium DTPA bovine serum albumin (BSA-DTPA-Gd) complex was injected i.v., followed by fixation in 10% formalin and removal from the animal. The sample was imaged at 9.4 T, yielding voxel sizes of 40 μm. Intravital microscopy was used to visualize the position and number of arterioles entering window chamber tumour preparations. Phosphorescence life time imaging (PLI) was used to measure vascular pO2. Blue and green light excitations of the upper and lower surfaces of window chambers were made (penetration depth of light ~50 vs >200 μm respectively). Arteriolar input into window chamber tumours was limited to 1 or 2 vessels, and appeared to be constrained to the fascial surface upon which the tumour grows. PLI of

  12. Development of a rapid method for the sequential extraction and subsequent quantification of fatty acids and sugars from avocado mesocarp tissue.

    PubMed

    Meyer, Marjolaine D; Terry, Leon A

    2008-08-27

    Methods devised for oil extraction from avocado (Persea americana Mill.) mesocarp (e.g., Soxhlet) are usually lengthy and require operation at high temperature. Moreover, methods for extracting sugars from avocado tissue (e.g., 80% ethanol, v/v) do not allow for lipids to be easily measured from the same sample. This study describes a new simple method that enabled sequential extraction and subsequent quantification of both fatty acids and sugars from the same avocado mesocarp tissue sample. Freeze-dried mesocarp samples of avocado cv. Hass fruit of different ripening stages were extracted by homogenization with hexane and the oil extracts quantified for fatty acid composition by GC. The resulting filter residues were readily usable for sugar extraction with methanol (62.5%, v/v). For comparison, oil was also extracted using the standard Soxhlet technique and the resulting thimble residue extracted for sugars as before. An additional experiment was carried out whereby filter residues were also extracted using ethanol. Average oil yield using the Soxhlet technique was significantly (P < 0.05) higher than that obtained by homogenization with hexane, although the difference remained very slight, and fatty acid profiles of the oil extracts following both methods were very similar. Oil recovery improved with increasing ripeness of the fruit with minor differences observed in the fatty acid composition during postharvest ripening. After lipid removal, methanolic extraction was superior in recovering sucrose and perseitol as compared to 80% ethanol (v/v), whereas mannoheptulose recovery was not affected by solvent used. The method presented has the benefits of shorter extraction time, lower extraction temperature, and reduced amount of solvent and can be used for sequential extraction of fatty acids and sugars from the same sample.

  13. 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.

  14. Prediction of recurrence by quantification of p185neu protein in non-small-cell lung cancer tissue.

    PubMed Central

    Diez, M.; Pollán, M.; Maestro, M.; Torres, A.; Ortega, D.; Gómez, A.; Sánchez, A.; Hernando, F.; Balibrea, J. L.

    1997-01-01

    The concentration of c-erbB-2 oncogene-encoded protein (p185neu) in fresh tumour samples obtained at the time of surgery from 94 non-small-cell lung cancer patients (NSCLC) was determined by an enzyme immunoassay. The relative prognostic importance was estimated, and the influence of other predictors was assessed by means of a Cox's proportional regression model. Median concentration of p185 in tumour tissues was 206 U mg(-1) (range 21-1050 U mg(-1)). p185 level did not differ significantly among subgroups defined by TNM classification, histological type, sex and age. Categorization of patients by p185 level, with 206 U mg(-1) and 343 U mg(-1) taken as cut-off values (corresponding to the 50th and 80th percentiles of the frequency distribution), showed that the recurrence rate, cumulative disease-free likelihood at the 36-month follow-up and median time from surgery to the diagnosis of recurrence worsened progressively as the level of p185 increased. Multivariate analysis confirmed the independent prognostic value of p185 level. Risk of recurrence increased by 1.304 for every increase of 100 units in p185 concentration (95% CI 1.141-1.490) (P<0.001). These findings encourage the inclusion of p185 concentration assay in a future predictive multifactorial prognostic index in NSCLC. PMID:9043025

  15. A simple, sensitive HPLC-PDA method for the quantification of itraconazole and hydroxy itraconazole in human serum: a reference laboratory experience.

    PubMed

    Cendejas-Bueno, Emilio; Cuenca-Estrella, Manuel; Gomez-Lopez, Alicia

    2013-07-01

    A chromatographic method (high-performance liquid chromatography/photodiode array detection) for the simultaneous determination of itraconazole and its mayor metabolite, hydroxy itraconazole, was developed. The validation data demonstrated acceptable values for linearity (dynamic range between 0.25-16 μg/mL for hydroxy itraconazole, 0.125-4 μg/mL for itraconazole), precision (Coefficient of Variation, 2.23-4.28% and 1.61-9.52% respectively), accuracy (Relative Error, -2.89 to -17.26% and -3.11 to 14.44%), selectivity, sensitivity (Lower limit of Quantification, 0.125 and 0.25 μg/mL), and recovery (97.30-105.82%) for both compounds. Its clinical suitability was also investigated in 104 trough human serum samples. The assay resulted to be rapid, sensitive, and simple for simultaneously quantification of these azole compounds in serum samples and should make a positive contribution to optimize therapies on an individual basis.

  16. 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.

  17. 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.

  18. 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

  19. 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. PMID:26428313

  20. 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.

  1. Expression stability of reference genes for quantitative RT-PCR of healthy and diseased pituitary tissue samples varies between humans, mice, and dogs.

    PubMed

    van Rijn, Sarah J; Riemers, Frank M; van den Heuvel, Douwe; Wolfswinkel, Jeannette; Hofland, Leo; Meij, Björn P; Penning, Louis C

    2014-04-01

    Pituitary surgery generates pituitary tissue for histology, immunohistochemistry, and molecular biological research. In the last decade, the pathogenesis of pituitary adenomas has been extensively studied in humans, and to a lesser degree in dogs, and tumor oncogenesis has been studied in knock-out mice, often by means of quantitative reversed-transcriptase PCR (RT-qPCR). A precondition of such analyses is that so-called reference genes are stably expressed regardless of changes in disease status or treatment. In this study, the expression of six frequently used reference genes, namely, tata box binding protein (tbp), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (ywhaz), hydroxymethylbilane synthase (hmbs), beta-2-microglobulin (b2m), succinate dehydrogenase complex subunit A (sdha), and glyceraldehyde 3 phosphate dehydrogenase 1 (gapdh), was studied in pituitary tissue (normal and adenoma) from three species (humans, mice, and dogs). The stability of expression of these reference genes differed between species and between healthy and diseased tissue within one species. Quantitative analysis based on a single reference gene that is assumed to be stably expressed might lead to wrong conclusions. This cross-species analysis clearly emphasizes the need to evaluate the expression stability of reference genes as a standard and integral aspect of study design and data analysis, in order to improve the validity of the conclusions drawn on the basis of quantitative molecular analyses.

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

    PubMed

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

    2014-06-01

    An automated solid-phase extraction (SPE) protocol followed by gas chromatography coupled with tandem mass spectrometry was developed for quantification of caffeine, cyamemazine, meprobamate, morphine and 6-monoacetylmorphine (6-MAM) in 11 biological matrices [blood, urine, bile, vitreous humor, liver, kidney, lung and skeletal muscle, brain, adipose tissue and bone marrow (BM)]. The assay was validated for linearity, within- and between-day precision and accuracy, limits of quantification, selectivity, extraction recovery (ER), sample dilution and autosampler stability on BM. For the other matrices, partial validation was performed (limits of quantification, linearity, within-day precision, accuracy, selectivity and ER). The lower limits of quantification were 12.5 ng/mL(ng/g) for 6-MAM, morphine and cyamemazine, 100 ng/mL(ng/g) for meprobamate and 50 ng/mL(ng/g) for caffeine. Analysis of real-case samples demonstrated the performance of the assay in forensic toxicology to investigate challenging cases in which, for example, blood is not available or in which analysis in alternative matrices could be relevant. The SPE protocol was also assessed as an extraction procedure that could target other relevant analytes of interest. The extraction procedure was applied to 12 molecules of forensic interest with various physicochemical properties (alimemazine, alprazolam, amitriptyline, citalopram, cocaine, diazepam, levomepromazine, nordazepam, tramadol, venlafaxine, pentobarbital and phenobarbital). All drugs were able to be detected at therapeutic concentrations in blood and in the alternate matrices. PMID:24790060

  3. Reference gene validation for quantification of gene expression during final oocyte maturation induced by diethylstilbestrol and di-(2-ethylhexyl)-phthalate in common carp.

    PubMed

    Shi, Yanyan; Lu, Jie; Wang, Yilei; Wang, Shuhong

    2016-08-01

    Final oocyte maturation is the key step to successful spawning and fertilization. Quantitative real-time PCR (qPCR) is the technique of election to quantify the abundance of functional genes in such study. Reference gene is essential for correct interpretation of qPCR data. However, an ideal universal reference gene that is stable under all experimental circumstances has not been described. Researchers should validate their reference genes while performing qPCR analysis. The expression of 6 candidate reference genes: 18s rRNA, 28s rRNA, Cathepsin Z, Elongation factor 1-α, Glyceraldehyde-3-phosphate dehydrogenase and β-actin were investigated during final oocyte maturation induced by different compounds (DES and DEHP) in common carp (Cyprinus carpio). Four softwares (Bestkeeper, geNorm, NormFinder and RefFinder) were used to screen the most stable gene in order to evaluate their expression stability. The results revealed that EF1α was highly stable expressed when final oocyte maturation was induced by DES, while gapdh was the most stable gene when final oocyte maturation was induced by DEHP. Stable expressed reference gene selection is critical for all qPCR analysis to get accurate target gene mRNA expression information. PMID:27521935

  4. Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms.

    PubMed

    Wang, Hou-Ling; Li, Lan; Tang, Sha; Yuan, Chao; Tian, Qianqian; Su, Yanyan; Li, Hui-Guang; Zhao, Lin; Yin, Weilun; Zhao, Rui; Xia, Xinli

    2015-01-01

    Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues.

  5. Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms

    PubMed Central

    Wang, Hou-Ling; Li, Lan; Tang, Sha; Yuan, Chao; Tian, Qianqian; Su, Yanyan; Li, Hui-Guang; Zhao, Lin; Yin, Weilun; Zhao, Rui; Xia, Xinli

    2015-01-01

    Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues. PMID:26343648

  6. 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.

  7. Simultaneous quantification of paeoniflorin, nobiletin, tangeretin, liquiritigenin, isoliquiritigenin, liquiritin and formononetin from Si-Ni-San extract in rat plasma and tissues by liquid chromatography-tandem mass spectrometry.

    PubMed

    Li, Tianxue; Yan, Zhixiang; Zhou, Chen; Sun, Jian; Jiang, Chuan; Yang, Xinghao

    2013-08-01

    In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of seven bioactive components including paeoniflorin, nobiletin, tangeretin, liquiritigenin, isoliquiritigenin, liquiritin and formononetin in rat plasma and tissues after oral administration of Si-Ni-San extract using astragaloside IV as internal standard (IS). The plasma and tissue samples were extracted by solid-phase extraction. Chromatographic separation was accomplished on a C18 column with a multiple-step gradient elution. The quantification was obtained by scanning with multiple reaction monitoring via an electrospray ionization source that was operated by switching between the positive and negative modes in two MS/MS scan segments. Full validation of the assay was implemented. In conclusion, this method demonstrated good linearity and specificity. The lower limits of quantification for the analytes were <7.5 ng/mL. Intra- and inter-day precisions (RSD) were <12.5% and accuracy (RE) ranged from -10.2 to 7.3%. The average recoveries of the analytes from rat plasma and tissues were >65.2% and 58.6%, respectively. The validated method was further applied to the determination of actual rat plasma and tissues after oral administration of Si-Ni-San extract. The results provided a meaningful basis for the clinical application of this prescription.

  8. 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.

  9. 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.

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

    PubMed

    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

  11. 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

  12. 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.

  13. 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.

  14. 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. PMID:20383095

  15. Automated Spatial Brain Normalization and Hindbrain White Matter Reference Tissue Give Improved [18F]-Florbetaben PET Quantitation in Alzheimer's Model Mice

    PubMed Central

    Overhoff, Felix; Brendel, Matthias; Jaworska, Anna; Korzhova, Viktoria; Delker, Andreas; Probst, Federico; Focke, Carola; Gildehaus, Franz-Josef; Carlsen, Janette; Baumann, Karlheinz; Haass, Christian; Bartenstein, Peter; Herms, Jochen; Rominger, Axel

    2016-01-01

    normalization with reference region templates presents an excellent method to avoid the inter-reader variability in preclinical Aβ-PET scans. Intracerebral reference regions lacking Aβ pathology serve for precise longitudinal in vivo quantification of [18F]-florbetaben PET. Hindbrain white matter reference performed best when considering the composite of quality criteria. PMID:26973442

  16. Some theoretical derivations relating to the tissue dosimetry of brachytherapy nuclides, with particular reference to iodine-125

    SciTech Connect

    Dale, R.G.

    1983-03-01

    Using a Monte Carlo computer technique, tables of parameters have been derived which are of use in the dosimetry of brachytherapy nuclides in a variety of tissues and organs. From the results, it has been possible to derive relationships linking the composition of a tissue with the dose received at any location around an implanted source. Other factors, such as the relative importance of scattered radiation, spectral degradation, and integral dose, are also discussed. In particular, attention is drawn to possible dosimetric problems arising from the clinical use of /sup 125/I.

  17. Differential distribution of U6 (RNU6-1) expression in human carcinoma tissues demonstrates the requirement for caution in the internal control gene selection for microRNA quantification.

    PubMed

    Lou, Ge; Ma, Ning; Xu, Ya; Jiang, Lei; Yang, Jing; Wang, Chuxuan; Jiao, Yufei; Gao, Xu

    2015-11-01

    Alterations in microRNA (miRNA) expression patterns have been associated with a number of human diseases. Accurate quantitation of miRNA levels is important for their use as biomarkers and in determining their functions. Although the issue of proper miRNA detection was solved with the introduction of standard reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) assays, numerous issues with the selection of appropriate internal control genes remain. U6 (RNU6‑1) snRNA, the most commonly used internal control gene in miRNA RT‑qPCR assays, was shown to be unstable in clinical samples, particularly cancer tissues. Identification of the distribution of U6 in different tissues is the premise of more accurate quantification of miRNAs. However, the distribution of U6 in human carcinoma tissues and corresponding normal tissues is unknown. In the present study, U6 levels were significantly higher in human breast carcinoma tissues compared with the corresponding normal tissues by RT‑qPCR. In the carcinoma or corresponding adjacent normal tissues, the expression levels of U6 in epithelial cells were higher than those in the mesenchymal cells. Furthermore, the expression levels of U6 in the carcinoma tissues of the liver and intrahepatic bile ducts were higher than those in the adjacent normal tissues. These results suggest that the expression and distribution of U6 exhibits a high degree of variability among several types of human cells. Therefore, caution is required when selecting U6 as an internal control gene for evaluating expression profiles of miRNAs in patients with carcinoma, particularly carcinoma of the liver and intrahepatic bile ducts.

  18. Evaluation of reference genes for quantitative polymerase chain reaction across life cycle stages and tissue types of Tribolium castaneum.

    PubMed

    Toutges, Michelle J; Hartzer, Kris; Lord, Jeffrey; Oppert, Brenda

    2010-08-25

    The genome of the genetic model for coleopteran insects, Tribolium castaneum, is now available for downstream applications. To facilitate gene expression studies in T. castaneum, genes were evaluated for suitability as normalizers in comparisons across tissues and/or developmental stages. In less diverse samples, such as comparisons within developmental stages or tissue only, normalizers for mRNA were more stable and consistent. Overall, the genes for ribosomal proteins rps6, rpl13a, rps3, and rps18 were the most stable normalizers for broad scale gene expression analysis in T. castaneum. However, their stability ranking was dependent upon the instrument as well as the analysis program. These data emphasize the need to optimize normalizers used in all real-time polymerase chain reaction experiments specifically for the experimental conditions and thermocycler and to carefully evaluate data generated by computational algorithms.

  19. Identification of reference genes for RT-qPCR in ovine mammary tissue during late pregnancy and lactation and in response to maternal nutritional programming.

    PubMed

    Paten, A M; Pain, S J; Peterson, S W; Blair, H T; Kenyon, P R; Dearden, P K; Duncan, E J

    2014-08-01

    The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland.

  20. 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.

  1. Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-dilution Method

    PubMed Central

    Fu, Lijuan; Amato, Nicolas J.; Wang, Pengcheng; McGowan, Sara J.; Niedernhofer, Laura J.; Wang, Yinsheng

    2016-01-01

    The rising interest in understanding the functions, regulation and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Mono-methylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m5C), 2′-O-methylcytidine (Cm), N6-methyladenosine (m6A) and 2′-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of the four methylated nucleosides are tissue-specific. In addition, the 2′-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m5C and m6A) in total RNA isolated from mouse brain, pancreas and spleen, but not mouse heart. We also found that the levels of m5C, Cm and Am are significantly lower (by 6.5-43 fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m6A was slightly higher (by 1.6 fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications. PMID:26158405

  2. 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.

  3. 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. PMID:26213073

  4. Estimation of drug receptor occupancy when non-displaceable binding differs between brain regions – extending the simplified reference tissue model

    PubMed Central

    Kågedal, Matts; Varnäs, Katarina; Hooker, Andrew C; Karlsson, Mats O

    2015-01-01

    Aim The simplified reference tissue model (SRTM) is used for estimation of receptor occupancy assuming that the non-displaceable binding in the reference region is identical to the brain regions of interest. The aim of this work was to extend the SRTM to also account for inter-regional differences in non-displaceable concentrations, and to investigate if this model allowed estimation of receptor occupancy using white matter as reference. It was also investigated if an apparent higher affinity in caudate compared with other brain regions, could be better explained by a difference in the extent of non-displaceable binding. Methods The analysis was based on a PET study in six healthy volunteers using the 5-HT1B receptor radioligand [11C]-AZ10419369. The radioligand was given intravenously as a tracer dose alone and following different oral doses of the 5-HT1B receptor antagonist AZD3783. Non-linear mixed effects models were developed where differences between regions in non-specific concentrations were accounted for. The properties of the models were also evaluated by means of simulation studies. Results The estimate (95% CI) of KiPL was 10.2 ng ml−1 (5.4, 15) and 10.4 ng ml−1 (8.1, 13.6) based on the extended SRTM with white matter as reference and based on the SRTM using cerebellum as reference, respectively. The estimate (95% CI) of KiPL for caudate relative to other brain regions was 55% (48, 62%). Conclusions The extended SRTM allows consideration of white matter as reference region when no suitable grey matter region exists. AZD3783 affinity appears to be higher in the caudate compared with other brain regions. PMID:25406494

  5. Validation of the Reference Tissue Model for Estimation of Dopaminergic D2-Like Receptor Binding With [18 F] (N-Methyl)Benperidol in Humans

    PubMed Central

    Antenor-Dorsey, Jo Ann V.; Markham, Joanne; Moerlein, Stephen M.; Videen, Tom O.; Perlmutter, Joel S.

    2008-01-01

    PET measurements of dopaminergic D2-like receptors may provide important insights into disorders such as Parkinson's disease, schizophrenia, dystonia and Tourette's syndrome. The PET radioligand [18 F] (N-Methyl)Benperidol ([18F]-NMB) has high affinity and selectivity for D2-like receptors and is not displaced by endogenous dopamine. The goal of this study is to evaluate use of a graphical method utilizing a reference tissue region for ([18F]-NMB PET analysis by comparisons to an explicit three-compartment tracer kinetic model and graphical method that use arterial blood measurements. We estimated binding potential (BP) in the caudate and putamen using all three methods in 16 humans and found that the three-compartment tracer kinetic method provided the highest BP estimates while the graphical method using a reference region yielded the lowest estimates (p<0.0001 by repeated measures ANOVA). However, the three methods yielded highly correlated BP estimates for the two regions of interest. We conclude that the graphical method using a reference region still provides a useful estimate of BP comparable to methods using arterial blood sampling, especially since the reference region method is less invasive and computationally more straightforward; thereby simplifying these measurements. PMID:18355689

  6. Photosynthetic carbon acquisition in Sargassum henslowianum (Fucales, Phaeophyta), with special reference to the comparison between the vegetative and reproductive tissues.

    PubMed

    Zou, Dinghui; Gao, Kunshan; Chen, Weizhou

    2011-02-01

    The photosynthetic oxygen evolution characteristics were examined in both vegetative (blade) and sexual reproductive (receptacle) tissues of Sargassum henslowianum (Fucales, Phaeophyta) from the Shenao bay of Nanao Island, China, to establish the mechanism of photosynthetic acquisition of inorganic carbon (Ci) in this species. In natural seawater (pH 8.1, ca. 2.2 mM Ci), irradiance-saturated net photosynthetic rate (NPR) was greater by 25.3% in blade than receptacle, whereas dark respiratory rate (DR) was 2-fold higher in receptacle than blade. NPR at pH 8.1 was nearly saturated with the 2.2 mM Ci for both blade and receptacle. However, the values of the half-saturation constant for Ci were sharply increased at pH 9.0. NPR was significantly affected, but DR was remained unchanged, with the variation of the pH values in seawater. The data from the final pH value derived from the pH-drift experiments and the comparison between the measured and theoretically estimated photosynthetic rates suggested that both blade and receptacle were capable of acquiring HCO(3)(-) in seawater. The inhibitors experiments showed that a HCO(3)(-) dehydration mechanism mediated by external carbonic anhydrase activity occurred in both the blade and receptacle tissues of S. henslowianum. The proton buffer TRIS had no inhibitory effect on NPR at normal pH value in natural seawater (pH 8.1), but it significantly depressed NPR at pH 9.0. This suggested that proton transport occurred at the outside of the plasma membrane facilitated the operation of the carbon acquisition at pH 9.0. It was proposed that the strategy of photosynthetic carbon acquisition at higher pH would prevent the alga from the damage of over-excitation and photoinhibition in case of sunshine and calm water. We concluded that the blade and receptacle tissues of S. henslowianum have similar mechanism of acquisition of exogenous Ci from seawater to drive photosynthesis; yet they are differentiated more or less with the

  7. Comparative assessment of parametric neuroreceptor mapping approaches based on the simplified reference tissue model using [¹¹C]ABP688 PET.

    PubMed

    Seo, Seongho; Kim, Su J; Kim, Yu K; Lee, Jee-Young; Jeong, Jae M; Lee, Dong S; Lee, Jae S

    2015-12-01

    In recent years, several linearized model approaches for fast and reliable parametric neuroreceptor mapping based on dynamic nuclear imaging have been developed from the simplified reference tissue model (SRTM) equation. All the methods share the basic SRTM assumptions, but use different schemes to alleviate the effect of noise in dynamic-image voxels. Thus, this study aimed to compare those approaches in terms of their performance in parametric image generation. We used the basis function method and MRTM2 (multilinear reference tissue model with two parameters), which require a division process to obtain the distribution volume ratio (DVR). In addition, a linear model with the DVR as a model parameter (multilinear SRTM) was used in two forms: one based on linear least squares and the other based on extension of total least squares (TLS). Assessment using simulated and actual dynamic [(11)C]ABP688 positron emission tomography data revealed their equivalence with the SRTM, except for different noise susceptibilities. In the DVR image production, the two multilinear SRTM approaches achieved better image quality and regional compatibility with the SRTM than the others, with slightly better performance in the TLS-based method. PMID:26243707

  8. 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

  9. 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

  10. Validation of a gas chromatography-ion trap-tandem mass spectrometry assay for the simultaneous quantification of cocaine, benzoylecgonine, cocaethylene, morphine, codeine, and 6-acetylmorphine in aqueous solution, blood, and skeletal muscle tissue.

    PubMed

    Rees, Kelly A; McLaughlin, Poppy A; Osselton, M David

    2012-01-01

    A gas chromatography-ion trap-tandem mass spectrometry method was developed and validated for the simultaneous solid-phase extraction and quantification of cocaine, benzoylecgonine, cocaethylene, morphine, codeine, and 6-monoacteylmorphine in blood, muscle tissue, and water. An assay for total morphine in blood and muscle was also validated. The limit of quantification was ≤ 0.01 mg/kg in muscle and ≤ 0.005 mg/L in blood and water. Good linearity was observed over the concentration ranges studied (r ≥ 0.99). The repeatability (% RSD) at the three concentration levels was typically ≤ 15% and never exceeded 17%. Intermediate precision of ≤ 16% was obtained for all matrices. Deviation < 20% of the nominal concentration was obtained in all repeatability and intermediate precision experiments. Extraction recoveries for cocaine and metabolites ranged from 91% to 110% in water, 81% to 110% in blood, and 61% to 75% in muscle. Recoveries for opiates ranged from 59% to 104% in water, 50% to 95% in blood, and 41% to 79% in muscle. The hydrolysis efficiency for the total morphine assay in blood and muscle ranged from 91% to 99% with within-day and intermediate precisions of ≤ 14% and ≤ 12%, respectively. PMID:22290746

  11. 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. PMID:27221202

  12. An improved method for absolute quantification of mRNA using multiplex polymerase chain reaction: determination of renin and angiotensinogen mRNA levels in various tissues.

    PubMed

    Dostal, D E; Rothblum, K N; Baker, K M

    1994-12-01

    We have developed a multiplex, competitive, reverse-transcriptase polymerase chain reaction (RT-PCR) method which measures absolute levels of renin, angiotensinogen, and the housekeeping transcript elongation factor-1 alpha (EF-1 alpha) mRNA. Sample RNA was simultaneously titrated with serial dilutions of renin, angiotensinogen, and EF-1 alpha competitor RNAs which flanked the endogenous concentrations of target transcripts. The samples were coreverse transcribed in the presence of random primers and resulting first-strand cDNA was coamplified for 10-15 cycles with [32P]-dCTP and primers for renin angiotensinogen, after which EF-1 alpha primers were added. Amplified DNA was separated by electrophoresis on polyacrylamide gel and radioactivity in the bands was quantified by direct radioanalytical scanning. Three conditions were necessary to obtain absolute quantification of renin and angiotensinogen mRNA levels: (a) exogenous competitor RNA was used to control for tube-to-tube variability in the efficiencies of reverse transcription and amplification; (b) Sample RNA was titrated with flanking concentrations of competitor RNA to correct for intraassay differences in the efficiency of amplification due to concentration differences between competitor and target templates; and (c) a housekeeping transcript EF-1 alpha was used to control for tube-to-tube differences in RNA loading and/or degradation. We show that the multiplex RT-PCR method is precise and accurate over approximately three logs of transcript concentration and sensitive to less than 5 and 0.5 fg for renin and angiotensinogen mRNA, respectively. This method will be useful for absolute quantification of target mRNAs, especially when the amount of sample RNA is limited or unknown and/or the gene expression is low. PMID:7887470

  13. 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)

  14. Quantification of Age-Related Tissue-Level Failure Strains of Rat Femoral Cortical Bones Using an Approach Combining Macrocompressive Test and Microfinite Element Analysis.

    PubMed

    Fan, Ruoxun; Gong, He; Zhang, Rui; Gao, Jiazi; Jia, Zhengbin; Hu, Yanjuan

    2016-04-01

    Bone mechanical properties vary with age; meanwhile, a close relationship exists among bone mechanical properties at different levels. Therefore, conducting multilevel analyses for bone structures with different ages are necessary to elucidate the effects of aging on bone mechanical properties at different levels. In this study, an approach that combined microfinite element (micro-FE) analysis and macrocompressive test was established to simulate the failure of male rat femoral cortical bone. Micro-FE analyses were primarily performed for rat cortical bones with different ages to simulate their failure processes under compressive load. Tissue-level failure strains in tension and compression of these cortical bones were then back-calculated by fitting the experimental stress-strain curves. Thus, tissue-level failure strains of rat femoral cortical bones with different ages were quantified. The tissue-level failure strain exhibited a biphasic behavior with age: in the period of skeletal maturity (1-7 months of age), the failure strain gradually increased; when the rat exceeded 7 months of age, the failure strain sharply decreased. In the period of skeletal maturity, both the macro- and tissue-levels mechanical properties showed a large promotion. In the period of skeletal aging (9-15 months of age), the tissue-level mechanical properties sharply deteriorated; however, the macromechanical properties only slightly deteriorated. The age-related changes in tissue-level failure strain were revealed through the analysis of male rat femoral cortical bones with different ages, which provided a theoretical basis to understand the relationship between rat cortical bone mechanical properties at macro- and tissue-levels and decrease of bone strength with age. PMID:26902102

  15. Quantification of Age-Related Tissue-Level Failure Strains of Rat Femoral Cortical Bones Using an Approach Combining Macrocompressive Test and Microfinite Element Analysis.

    PubMed

    Fan, Ruoxun; Gong, He; Zhang, Rui; Gao, Jiazi; Jia, Zhengbin; Hu, Yanjuan

    2016-04-01

    Bone mechanical properties vary with age; meanwhile, a close relationship exists among bone mechanical properties at different levels. Therefore, conducting multilevel analyses for bone structures with different ages are necessary to elucidate the effects of aging on bone mechanical properties at different levels. In this study, an approach that combined microfinite element (micro-FE) analysis and macrocompressive test was established to simulate the failure of male rat femoral cortical bone. Micro-FE analyses were primarily performed for rat cortical bones with different ages to simulate their failure processes under compressive load. Tissue-level failure strains in tension and compression of these cortical bones were then back-calculated by fitting the experimental stress-strain curves. Thus, tissue-level failure strains of rat femoral cortical bones with different ages were quantified. The tissue-level failure strain exhibited a biphasic behavior with age: in the period of skeletal maturity (1-7 months of age), the failure strain gradually increased; when the rat exceeded 7 months of age, the failure strain sharply decreased. In the period of skeletal maturity, both the macro- and tissue-levels mechanical properties showed a large promotion. In the period of skeletal aging (9-15 months of age), the tissue-level mechanical properties sharply deteriorated; however, the macromechanical properties only slightly deteriorated. The age-related changes in tissue-level failure strain were revealed through the analysis of male rat femoral cortical bones with different ages, which provided a theoretical basis to understand the relationship between rat cortical bone mechanical properties at macro- and tissue-levels and decrease of bone strength with age.

  16. 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.

  17. 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

    2015-06-01

    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. PMID:25992999

  18. PCA3 gene expression in prostate cancer tissue in a Chinese population: quantification by real-time FQ-RT-PCR based on exon 3 of PCA3.

    PubMed

    Tao, Zhihua; Shen, Mo; Zheng, Yanbo; Mao, Xiaolu; Chen, Zhanguo; Yin, Yibing; Yu, Kaiyuan; Weng, Zhiliang; Xie, Hui; Li, Chengdi; Wu, Xiuling; Hu, Yuanping; Zhang, Xiaohua; Wang, Ouchen; Song, Qitong; Yu, Zhixian

    2010-08-01

    Prostate cancer (PCa) is the second most common cancer in men, and its incidence is still increasing. PCA3 is the most prostate cancer specific biomarker. Here we confirmed that both exon 3 and exon 4 are in the prostate-specific region of the PCA3 gene, and established the methodology of real-time fluorescent quantitative RT-PCR (FQ-RT-PCR) detecting PCA3 mRNA with primer spanning exons 1 and 3, and evaluated its clinical utility in a Chinese population. What disclosed that PCA3 mRNA is prostate cancer specific and shows increased expression in prostate cancer. It could be a reliable molecular marker in prostate cancer diagnosis. Exon 3-based real-time FQ-RT-PCR may prove useful in prostate cancer diagnosis, given that the associated primer would span only exons 1 and 3, relative to other models spanning exons 1 to 4. A shorter amplicon would not only enhance the efficiency of real-time FQ-RT-PCR, but may also simplify the quantification of PCA3 mRNA.

  19. 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. PMID:26713090

  20. 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

  1. Quantification of the global and local complexity of the epithelial-connective tissue interface of normal, dysplastic, and neoplastic oral mucosae using digital imaging.

    PubMed

    Abu Eid, Rasha; Landini, Gabriel

    2003-01-01

    This study aimed at quantifying the complexity of the epithelial-connective tissue interface (ECTI) in human normal mucosa, premalignant, and malignant lesions using fractal geometry. Two approaches were used to describe the complexity of 377 oral mucosa ECTI profiles. The box counting method was used to estimate their global fractal dimension, while local fractal dimensions were estimated using the mass radius relation at various local scales. The ECTI complexity significantly increased from normal through premalignant to malignant profiles in both global and local (over 283 microm) scales. Normal mucosa samples from different sites of the oral cavity also had different degrees of global complexity. Fractal geometry is a useful morphological marker of tissue complexity changes taking place during epithelial malignancy and premalignancy, and we propose it as a quantitative marker of epithelial complexity. PMID:14521264

  2. 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.

  3. 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.

  4. 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

  5. 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

    2016-01-01

    (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. PMID:26902733

  6. In Vivo Quantification of 5-HT2A Brain Receptors in Mdr1a KO Rats with 123I-R91150 Single-Photon Emission Computed Tomography.

    PubMed

    Dumas, Noé; Moulin-Sallanon, Marcelle; Fender, Pascal; Tournier, Benjamin B; Ginovart, Nathalie; Charnay, Yves; Millet, Philippe

    2015-01-01

    Our goal was to identify suitable image quantification methods to image 5-hydroxytryptamine2A (5-HT2A) receptors in vivo in Mdr1a knockout (KO) rats (i.e., P-glycoprotein KO) using 123I-R91150 single-photon emission computed tomography (SPECT). The 123I-R91150 binding parameters estimated with different reference tissue models (simplified reference tissue model [SRTM], Logan reference tissue model, and tissue ratio [TR] method) were compared to the estimates obtained with a comprehensive three-tissue/seven-parameter (3T/7k)-based model. The SRTM and Logan reference tissue model estimates of 5-HT2A receptor (5-HT2AR) nondisplaceable binding potential (BPND) correlated well with the absolute receptor density measured with the 3T/7k gold standard (r > .89). Quantification of 5-HT2AR using the Logan reference tissue model required at least 90 minutes of scanning, whereas the SRTM required at least 110 minutes. The TR method estimates were also highly correlated to the 5-HT2AR density (r > .91) and only required a single 20-minute scan between 100 and 120 minutes postinjection. However, a systematic overestimation of the BPND values was observed. The Logan reference tissue method is more convenient than the SRTM for the quantification of 5-HT2AR in Mdr1a KO rats using 123I-R91150 SPECT. The TR method is an interesting and simple alternative, despite its bias, as it still provides a valid index of 5-HT2AR density. PMID:26105563

  7. 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

  8. 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

  9. In situ quantification of HER2–protein tyrosine kinase 6 (PTK6) protein–protein complexes in paraffin sections from breast cancer tissues

    PubMed Central

    Aubele, M; Spears, M; Ludyga, N; Braselmann, H; Feuchtinger, A; Taylor, K J; Lindner, K; Auer, G; Stering, K; Höfler, H; Schmitt, M; Bartlett, J M S

    2010-01-01

    Background: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. Method and results: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6–HER2 protein–protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). Conclusion: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2–PTK6 complexes are of prognostic relevance. PMID:20700126

  10. Histological characterization and quantification of cellular events following neural and fibroblast(-like) stem cell grafting in healthy and demyelinated CNS tissue.

    PubMed

    Praet, Jelle; Santermans, Eva; Reekmans, Kristien; de Vocht, Nathalie; Le Blon, Debbie; Hoornaert, Chloé; Daans, Jasmijn; Goossens, Herman; Berneman, Zwi; Hens, Niel; Van der Linden, Annemie; Ponsaerts, Peter

    2014-01-01

    Preclinical animal studies involving intracerebral (stem) cell grafting are gaining popularity in many laboratories due to the reported beneficial effects of cell grafting on various diseases or traumata of the central nervous system (CNS). In this chapter, we describe a histological workflow to characterize and quantify cellular events following neural and fibroblast(-like) stem cell grafting in healthy and demyelinated CNS tissue. First, we provide standardized protocols to isolate and culture eGFP(+) neural and fibroblast(-like) stem cells from embryonic mouse tissue. Second, we describe flow cytometric procedures to determine cell viability, eGFP transgene expression, and the expression of different stem cell lineage markers. Third, we explain how to induce reproducible demyelination in the CNS of mice by means of cuprizone administration, a validated mouse model for human multiple sclerosis. Fourth, the technical procedures for cell grafting in the CNS are explained in detail. Finally, an optimized and validated workflow for the quantitative histological analysis of cell graft survival and endogenous astroglial and microglial responses is provided. PMID:25173390

  11. Quantification of Oxidized and Unsaturated Bile Alcohols in Sea Lamprey Tissues by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Li, Ke; Scott, Anne M; Chung-Davidson, Yu-Wen; Bussy, Ugo; Patel, Trinkal; Middleton, Zoe E; Li, Weiming

    2016-01-01

    A sensitive and reliable method was developed and validated for the determination of unsaturated bile alcohols in sea lamprey tissues using liquid-liquid extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The liver, kidney, and intestine samples were extracted with acetonitrile and defatted by n-hexane. Gradient UHPLC separation was performed using an Acquity BEH C18 column with a mobile phase of water and methanol containing 20 mM triethylamine. Multiple reaction monitoring modes of precursor-product ion transitions for each analyte was used. This method displayed good linearity, with correlation coefficients greater than 0.99, and was validated. Precision and accuracy (RSD %) were in the range of 0.31%-5.28%, while mean recoveries were between 84.3%-96.3%. With this technique, sea lamprey tissue samples were analyzed for unsaturated bile alcohol analytes. This method is practical and particularly suitable for widespread putative pheromone residue analysis. PMID:27563866

  12. [Reference ranges of matrix metalloproteinase-1, -2, -9 and tissue inhibitor of matrix metalloproteinases-1 concentrations in amniotic fluid in physiological pregnancy].

    PubMed

    Korenovsky, Yu V; Remneva, O V

    2016-01-01

    The aim of this study was to determine reference values of matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-9 and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in the amniotic fluid at the first stage of labor in physiological pregnancy. 89 women at the first stage of term labor have been examined. Samples of amniotic fluid were taken at the first period of labor by vaginal amniotomy. Concentrations ofMMP-1, MMP-2, MMP-9, and TIMP-1 were investigated in amniotic fluid by ELISA kits. We have determined normal concentration ranges for MMP-1, MMP-2, MMP-9, TIMP-1, and ratios of concentrations of MMPs and TIMP-1 (MMP-1/TIMP-1, MMP-2/TIMP-1, MMP-9/TIMP-1) in the amniotic fluid at the first period of labor in physiological pregnancy. These included: MMP-1--5.1-16.8 pg/mg of protein, MMP-2--238.3-374.1 pg/mg of protein, MMP-9--66.1-113.3 pg/mg of protein, TIMP-1--4.7-13.6 pg/mg of protein, ratio of MMP-1/TIMP-1--0.1-2.2, ratio of MMP-2/TIMP-1--19.9-55.7, ratio of MMP-9/TIMP-1--4.2-17.2.

  13. A novel RT-PCR method for quantification of human papillomavirus transcripts in archived tissues and its application in oropharyngeal cancer prognosis.

    PubMed

    Gao, Ge; Chernock, Rebecca D; Gay, Hiram A; Thorstad, Wade L; Zhang, Tian R; Wang, Hongwei; Ma, Xiao-Jun; Luo, Yuling; Lewis, James S; Wang, Xiaowei

    2013-02-15

    Oropharyngeal squamous cell carcinoma (SCC) is strongly associated with human papillomavirus (HPV) infection, which is distinctively different from most other head and neck cancers. However, a robust quantitative reverse transcription PCR (RT-qPCR) method for comprehensive expression profiling of HPV genes in routinely fixed tissues has not been reported. To address this issue, we have established a new real-time RT-PCR method for the expression profiling of the E6 and E7 oncogenes from 13 high-risk HPV types. This method was validated in cervical cancer and by comparison with another HPV RNA detection method (in situ hybridization) in oropharyngeal tumors. In addition, the expression profiles of selected HPV-related human genes were also analyzed. HPV E6 and E7 expression profiles were then analyzed in 150 archived oropharyngeal SCC samples and compared with other variables and with patient outcomes. Our study showed that RT-qPCR and RNA in situ hybridization were 100% concordant in determining HPV status. HPV transcriptional activity was found in most oropharyngeal SCC (81.3%), a prevalence that is higher than in previous studies. Besides HPV16, three other HPV types were also detected, including 33, 35 and 18. Furthermore, HPV and p16 had essentially identical expression signatures, and both HPV and p16 were prognostic biomarkers for the prediction of disease outcome. Thus, p16 mRNA or protein expression signature is a sensitive and specific surrogate marker for HPV transcriptional activity (all genotypes combined).

  14. 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

  15. 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

  16. 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

  17. Quantification of endogenous brassinosteroids in sub-gram plant tissues by in-line matrix solid-phase dispersion-tandem solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Wang, Lu; Duan, Chunfeng; Wu, Dapeng; Guan, Yafeng

    2014-09-12

    A matrix solid-phase dispersion (MSPD)-tandem mixed mode anion exchange (MAX)-mixed mode cation exchange (MCX) solid phase extraction-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed for quantification of six endogenous brassinosteroids (BRs) (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, dolichosterone, teasterone and typhasterol) in rice plant tissues. Non-polar interferences were removed effectively by C8 dispersant used in MSPD, while the following tandem MAX-MCX process facilitated the elimination of polar and ionizable compounds. The weak reversed-phase retention feature of MAX-MCX leaded to good compatibility of the elution solvents in the in-line coupled MSPD-MAX-MCX system. This system was optimized for extraction and purification of BRs in plant samples. The effects of the type of solid phase, the elution solvent, the extraction temperature and the clean-up material were studied. Before HPLC separation, BRs purified were derivatized by m-aminophenylboronic acid to enhance the sensitivity of MS/MS to BRs. Compared with traditional liquid-liquid extraction and solid phase extraction (LLE-SPE), the proposed MSPD-MAX-MCX method showed higher extraction efficiency, lower matrix effect, and advantages of easy manipulation and time-saving. The in-line MSPD-MAX-MCX coupled with HPLC-MS/MS method provided a linear response over two orders of magnitude of BRs concentration with correlation coefficients above 0.9982, limits of detection between 0.008 and 0.04ngmL(-1), relative standard deviations (RSDs) below 29.4%, and recoveries above 77.8%. The proposed method has been successfully applied to analysis of endogenous BRs in rice plant at booting stage and maturity stage.

  18. 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

  19. 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.

  20. Infection studies with two highly pathogenic avian influenza strains (Vietnamese and Indonesian) in Pekin ducks (Anas platyrhynchos), with particular reference to clinical disease, tissue tropism and viral shedding.

    PubMed

    Bingham, John; Green, Diane J; Lowther, Sue; Klippel, Jessica; Burggraaf, Simon; Anderson, Danielle E; Wibawa, Hendra; Hoa, Dong Manh; Long, Ngo Thanh; Vu, Pham Phong; Middleton, Deborah J; Daniels, Peter W

    2009-08-01

    Pekin ducks were infected by the mucosal route (oral, nasal, ocular) with one of two strains of Eurasian lineage H5N1 highly pathogenic avian influenza virus: A/Muscovy duck/Vietnam/453/2004 and A/duck/Indramayu/BBVW/109/2006 (from Indonesia). Ducks were killed humanely on days 1, 2, 3, 5 and 7 after challenge, or whenever morbidity was severe enough to justify euthanasia. Morbidity was recorded by observation of clinical signs and cloacal temperatures; the disease was characterized by histopathology; tissue tropism was studied by immunohistochemistry and virus titration on tissue samples; and viral shedding patterns were determined by virus isolation and titration of oral and cloacal swabs. The Vietnamese strain caused severe morbidity with fever and depression; the Indonesian strain caused only transient fever. Both viruses had a predilection for a similar range of tissue types, but the quantity of tissue antigen and tissue virus titres were considerably higher with the Vietnamese strain. The Vietnamese strain caused severe myocarditis and skeletal myositis; both strains caused non-suppurative encephalitis and a range of other inflammatory reactions of varying severity. The principal epithelial tissue infected was that of the air sacs, but antigen was not abundant. Epithelium of the turbinates, trachea and bronchi had only rare infection with virus. Virus was shed from both the oral and cloacal routes; it was first detected 24 h after challenge and persisted until day 5 after challenge. The higher prevalence of virus from swabs from ducks infected with the Vietnamese strain indicates that this strain may be more adapted to ducks than the Indonesia strain.

  1. Assessment and quantification of patient set-up errors in nasopharyngeal cancer patients and their biological and dosimetric impact in terms of generalized equivalent uniform dose (gEUD), tumour control probability (TCP) and normal tissue complication probability (NTCP)

    PubMed Central

    Marcie, S; Fellah, M; Chami, S; Mekki, F

    2015-01-01

    Objective: The aim of this study is to assess and quantify patients' set-up errors using an electronic portal imaging device and to evaluate their dosimetric and biological impact in terms of generalized equivalent uniform dose (gEUD) on predictive models, such as the tumour control probability (TCP) and the normal tissue complication probability (NTCP). Methods: 20 patients treated for nasopharyngeal cancer were enrolled in the radiotherapy–oncology department of HCA. Systematic and random errors were quantified. The dosimetric and biological impact of these set-up errors on the target volume and the organ at risk (OARs) coverage were assessed using calculation of dose–volume histogram, gEUD, TCP and NTCP. For this purpose, an in-house software was developed and used. Results: The standard deviations (1SDs) of the systematic set-up and random set-up errors were calculated for the lateral and subclavicular fields and gave the following results: ∑ = 0.63 ± (0.42) mm and σ = 3.75 ± (0.79) mm, respectively. Thus a planning organ at risk volume (PRV) margin of 3 mm was defined around the OARs, and a 5-mm margin used around the clinical target volume. The gEUD, TCP and NTCP calculations obtained with and without set-up errors have shown increased values for tumour, where ΔgEUD (tumour) = 1.94% Gy (p = 0.00721) and ΔTCP = 2.03%. The toxicity of OARs was quantified using gEUD and NTCP. The values of ΔgEUD (OARs) vary from 0.78% to 5.95% in the case of the brainstem and the optic chiasm, respectively. The corresponding ΔNTCP varies from 0.15% to 0.53%, respectively. Conclusion: The quantification of set-up errors has a dosimetric and biological impact on the tumour and on the OARs. The developed in-house software using the concept of gEUD, TCP and NTCP biological models has been successfully used in this study. It can be used also to optimize the treatment plan established for our patients. Advances in knowledge: The g

  2. 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.

  3. Organochlorine pesticide levels in maternal blood and placental tissue with reference to preterm birth: a recent trend in North Indian population.

    PubMed

    Tyagi, Vipin; Garg, Neha; Mustafa, M D; Banerjee, B D; Guleria, Kiran

    2015-07-01

    Organochlorine pesticides (OCPs) have been widely used in public health and agriculture programs in developed as well as developing countries, including India. Being xenoestrogenic in nature, OCPs may act as endocrine disruptors leading to preterm birth (PTB) through disturbance of normal estrogen-progesterone ratio. PTB is the leading cause of neonatal deaths worldwide. Therefore, the present study is aimed to determine the extent to which persistent environmental chemicals may accumulate in pregnant women and placenta and ascertain possible associations between exposure level and period of gestation (POG), baby weight, and/or placental weight in PTB cases. Maternal blood and placenta samples of PTB cases (n = 50) and subjects of term delivery as controls (n = 50) were collected. OCP residue levels were estimated by the gas chromatography system equipped with an electron capture detector. Significantly higher levels of α-hexachlorocyclohexane (α-HCH), β-hexachlorocyclohexane (β-HCH), dichlorodiphenyldichloroethane (DDD), and dichlorodiphenyldichloroethylene (DDE) were found in maternal blood of PTB cases as compared to control. Significantly higher levels of DDE and dichlorodiphenyltrichloroethane (DDT) were also found in placental tissue of PTB cases as compared to control group. There was a statistically significant negative correlation between maternal blood level of α-HCH and birth-weight (r = -0.299) and POG (r = -0.234). γ-Hexachlorocyclohexane (γ-HCH) and dieldrin had a negative correlation with placental weight (r = -0.401 and -0.256, respectively), and DDE and β-HCH had a negative correlation with POG (r = -0.251 and -0.229, respectively). The presence of OCPs in maternal blood and placental tissue represents prenatal exposure hazard for fetuses due to chronic bioaccumulation and poor elimination with possible deleterious effect on health.

  4. Pneumothorax as adverse event in patients with lung metastases of soft tissue sarcoma treated with pazopanib: a single reference centre case series

    PubMed Central

    2014-01-01

    Background Recently, the phase III PALETTE study introduced pazopanib (Votrient®) as treatment for adult patients with locally advanced or metastatic non-liposarcoma soft tissue sarcoma after prior treatment with doxorubicin and/or ifosfamide. Pneumothorax was reported as adverse event in 8 of 246 treated patients (3.3%) in that study. This case series presents the incidence and clinic of this complication in the Leiden University Medical Centre. Cases Forty-three patients were treated with pazopanib of which six patients (14.0%) developed a pneumothorax. These six patients were treated for malignant peripheral nerve sheath tumour, angiosarcoma, synovial sarcoma, fibromyxomatoid sarcoma, pleomorphic sarcoma and endometrial stromal sarcoma. All six patients had subpleural pulmonary or pleural metastases at the start of pazopanib and the pneumothorax developed during or shortly after treatment with pazopanib and was difficult to treat. Discussion The incidence reported by us is higher than the incidence in the PALETTE study. Trials with pazopanib in renal cell carcinoma, urothelial carcinoma and cervix carcinoma did not report pneumothorax as an adverse event, suggesting pneumothorax as a specific adverse event in soft tissue sarcoma patients treated with pazopanib. This may be related to the fact that there is often pleural metastatic involvement and cystic degeneration due to pazopanib treatment may add to the risk. Conclusion The risk of an, often difficult to treat, pneumothorax during pazopanib therapy should be discussed with the patient before initiation of treatment for a pulmonary metastasized sarcoma and physicians should be alert to the occurrence of such an event. PMID:25302110

  5. Quantification of Secondary Metabolites.

    PubMed

    2016-01-01

    Plants are a rich source of secondary metabolites that have medicinal and aromatic properties. Secondary metabolites such as alkaloids, iridoids and phenolics generally produced by plants for their defence mechanisms have been implicated in the therapeutic properties of most medicinal plants. Hence, quantification of these metabolites will aid to discover new and effective drugs from plant sources and also to scientifically validate the existing traditional practices. Quantification of large group of phytochemicals such as phenolics and flavonoids is quantified in this context.

  6. 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

  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. Dehydroepiandrosterone sulfate quantification in serum using high-performance liquid chromatography/mass spectrometry and a deuterated internal standard: a technique suitable for routine use or as a reference method

    SciTech Connect

    Shackleton, C.H.; Kletke, C.; Wudy, S.; Pratt, J.H. )

    1990-10-01

    A thermospray high-performance liquid chromatography/mass spectrometry method for determination of serum dehydroepiandrosterone sulfate is described. The steroid was measured intact using (7,7-2H2)dehydroepiandrosterone sulfate as internal standard. The analysis was carried out in the negative ion mode by determining the peak height ratio of the molecular anions of the analyte and internal standard. The method was used to determine the steroid in serum from 15 male and female normal adults and the following values were obtained: males, 272 +/- 45 micrograms/dl (range, 197 to 331 micrograms/dl) and females, 215 +/- 67 micrograms/dl (range, 107 to 347 micrograms/dl). In addition, dehydroepiandrosterone sulfate was measured by high-performance liquid chromatography/mass spectrometry and radioimmunoassay (a commercial kit) on 25 individuals of all age groups. There was strong correlation between the values obtained, but the radioimmunoassay values were generally double those obtained by high-performance liquid chromatography/mass spectrometry. Three other steroid sulfates, androsterone sulfate, epiandrosterone sulfate, and androst-5-ene-3 beta, 17 beta-diol sulfate, were also assayed. In males, these had mean values of 112, 44, and 13 micrograms/dl and, in females, they had mean values of 84, 25, and 6 micrograms/dl, respectively. Radioimmunoassay cross-reactivity measurement for these steroids (as reference compounds) showed that they were unlikely to contribute greatly to the discrepancy between radioimmunoassay and high-performance liquid chromatography/mass spectrometry values.

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

    PubMed

    Hu, Yanting; 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.

  10. 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

  11. Image-based Tissue Distribution Modeling for Skeletal Muscle Quality Characterization

    PubMed Central

    Fishbein, K. W.; Moore, A. Z.; Spencer, R. G.; Ferrucci, L.

    2016-01-01

    The identification and characterization of regional body tissues is essential to understand changes that occur with aging and age-related metabolic diseases such as diabetes and obesity and how these diseases affect trajectories of health and functional status. Imaging technologies are frequently used to derive volumetric, area, and density measurements of different tissues. Despite the significance and direct applicability of automated tissue quantification and characterization techniques, these topics have remained relatively under-explored in the medical image analysis literature. We present a method for identification and characterization of muscle and adipose tissue in the mid-thigh region using MRI. We propose an image-based muscle quality prediction technique that estimates tissue-specific probability density models and their eigenstructures in the joint domain of water- and fat-suppressed voxel signal intensities along with volumetric and intensity-based tissue characteristics computed during the quantification stage. We evaluated the predictive capability of our approach against reference biomechanical muscle quality measurements using statistical tests and classification performance experiments. The reference standard for muscle quality is defined as the ratio of muscle strength to muscle mass. The results show promise for the development of non-invasive image-based muscle quality descriptors. PMID:26336111

  12. Selection of Reliable Reference Genes for Gene Expression Studies Using Real-Time PCR in Tung Tree during Seed Development

    PubMed Central

    Han, Xiaojiao; Lu, Mengzhu; Chen, Yicun; Zhan, Zhiyong; Cui, Qinqin; Wang, Yangdong

    2012-01-01

    Quantitative real-time PCR (RT-qPCR) has become an accurate and widely used technique to analyze expression levels of selected genes. It is very necessary to select appropriate reference genes for gene expression normalization. In the present study, we assessed the expression stability of 11 reference genes including eight traditional housekeeping genes and three novel genes in different tissues/organs and developing seeds from four cultivars of tung tree. All 11 reference genes showed a wide range of Ct values in all samples, indicating that they differently expressed. Three softwares – geNorm, NormFinder and BestKeeper – were used to determine the stability of these references except for ALB (2S albumin), which presented a little divergence. The results from the three softwares showed that ACT7 (Actin7a), UBQ (Ubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and EF1α (elongation factor 1-α) were the most stable reference genes across all of the tested tung samples and tung developing seeds, while ALB (2S albumin) was unsuitable as internal controls. ACT7, EF1β (elongation factor1-beta), GAPDH and TEF1 (transcription elongation factor 1) were the top four choices for different tissues/organs whereas LCR69 did not favor normalization of RT-qPCR in these tissues/organs. Meanwhile, the expression profiles of FAD2 and FADX were realized using stable reference genes. The relative quantification of the FAD2 and FADX genes varied according to the internal controls and the number of internal controls. The results further proved the importance of the choice of reference genes in the tung tree. These stable reference genes will be employed in normalization and quantification of transcript levels in future expression studies of tung genes. PMID:22912794

  13. 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"…

  14. 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…

  15. Quantification of nonclassicality

    NASA Astrophysics Data System (ADS)

    Gehrke, C.; Sperling, J.; Vogel, W.

    2012-11-01

    To quantify single-mode nonclassicality, we start from an operational approach. A positive semidefinite observable is introduced to describe a measurement setup. The quantification is based on the negativity of the normally ordered version of this observable. Perfect operational quantumness corresponds to the quantum-noise-free measurement of the chosen observable. Surprisingly, even moderately squeezed states may exhibit perfect quantumness for a properly designed measurement. The quantification is also considered from an axiomatic viewpoint, based on the algebraic structure of the quantum states and the quantum superposition principle. Basic conclusions from both approaches are consistent with this fundamental principle of the quantum world.

  16. Quantification of Secondary Metabolites.

    PubMed

    2016-01-01

    Plants are a rich source of secondary metabolites that have medicinal and aromatic properties. Secondary metabolites such as alkaloids, iridoids and phenolics generally produced by plants for their defence mechanisms have been implicated in the therapeutic properties of most medicinal plants. Hence, quantification of these metabolites will aid to discover new and effective drugs from plant sources and also to scientifically validate the existing traditional practices. Quantification of large group of phytochemicals such as phenolics and flavonoids is quantified in this context. PMID:26939265

  17. 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.

  18. 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

  19. Test-retest stability of cerebral mGluR₅ quantification using [¹¹C]ABP688 and positron emission tomography in rats.

    PubMed

    Elmenhorst, David; Aliaga, Antonio; Bauer, Andreas; Rosa-Neto, Pedro

    2012-06-01

    This study evaluates the reproducibility of the quantification of metabotropic glutamate receptor type 5 (mGluR₅) densities in rats using the PET radiotracer [¹¹C]ABP688 and pharmacokinetic models that are based on an input function, which is derived from a reference tissue. Seven rats underwent dynamic PET scans (60 min) after bolus injection of [¹¹C]ABP688. Kinetic analyses included: binding potential (BP(ND) ) determined by calculating (a) the simplified reference tissue model (SRTM) and (b) its two-steps simplified version (SRTM2); (c) multilinear reference tissue model (MRTM) and (d) its 2-parameter version (MRTM2); (e) noninvasive graphical analysis (NIGA). Parametric images were generated representing BP(ND) by the MRTM2 model. BP(ND) determinations were reproducible with low to acceptable variability ranging from 5 to 10% and reproducibility scores (intraclass correlation coefficient) between 0.51 and 0.88. The pharmacokinetic model that showed lowest overall variability was the SRTM. In contrast, the use of the NIGA was associated with significantly lower reproducibility scores. Comparison of parametric images revealed no significant bias between test and retest measurements and is therefore suitable to compare groups at voxel levels. In conclusion, our results suggest that noninvasive quantification of [¹¹C]ABP688 imaging is reproducible and reliable for PET studies of the cerebral mGluR₅ in rats. PMID:22290765

  20. 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)

  1. Quantification of GPCR mRNA using real-time RT-PCR.

    PubMed

    Brattelid, Trond; Levy, Finn Olav

    2011-01-01

    Characterisation of G-protein-coupled receptor (GPCR) mRNA expression under normal, different pharmacological and pathological conditions in experimental animal models and human tissue biopsies by quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a valuable approach to understand the regulation of GPCR expression. RT-qPCR is specific and sensitive with a broad dynamic range, which allows precise quantification of mRNA species of interest. In addition to measuring the relative levels of mRNA in a tissue or changes in expression levels between groups of genes of interest, RT-qPCR is also used to identify splice variants and single nucleotide polymorphisms (SNPs) of GPCRs. Even though RT-qPCR has become the standard method for quantification of gene expression, RT-qPCR is sensitive to RNA quality, assay design, normalisation approach and data analysis. This protocol is meant as a guide to RT-qPCR methodology with references to the best standard methods available at present.

  2. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-01

    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.

  3. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-01

    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. PMID:25411902

  4. Reference Materials

    NASA Astrophysics Data System (ADS)

    Merkus, Henk G.

    Reference materials for measurement of particle size and porosity may be used for calibration or qualification of instruments or for validation of operating procedures or operators. They cover a broad range of materials. On the one hand there are the certified reference materials, for which governmental institutes have certified one or more typical size or porosity values. Then, there is a large group of reference materials from commercial companies. And on the other hand there are typical products in a given line of industry, where size or porosity values come from the analysis laboratory itself or from some round-robin test in a group of industrial laboratories. Their regular application is essential for adequate quality control of particle size and porosity measurement, as required in e.g., ISO 17025 on quality management. In relation to this, some quality requirements for certification are presented.

  5. Quantificational logic of context

    SciTech Connect

    Buvac, Sasa

    1996-12-31

    In this paper we extend the Propositional Logic of Context, to the quantificational (predicate calculus) case. This extension is important in the declarative representation of knowledge for two reasons. Firstly, since contexts are objects in the semantics which can be denoted by terms in the language and which can be quantified over, the extension enables us to express arbitrary first-order properties of contexts. Secondly, since the extended language is no longer only propositional, we can express that an arbitrary predicate calculus formula is true in a context. The paper describes the syntax and the semantics of a quantificational language of context, gives a Hilbert style formal system, and outlines a proof of the system`s completeness.

  6. The proteomics quantification dilemma.

    PubMed

    Jungblut, Peter R

    2014-07-31

    Proteomics is dominated today by the protein expression discourse, which favorites the bottom-up approach because of its high throughput and its high sensitivity. For quantification this proceeding is misleading, if a protein is present with more than one protein species in the sample to be analyzed. The protein speciation discourse considers this more realistic situation and affords the top-down procedures or at least a separation of the protein species in advance to identification and quantification. Today all of the top-down procedures are one order of magnitude less sensitive than the bottom-up ones. To increase sensitivity and to increase throughput are major challenges for proteomics of the next years. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez. PMID:24681132

  7. 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

  8. Validation of Suitable Reference Genes for Assessing Gene Expression of MicroRNAs in Lonicera japonica.

    PubMed

    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 (C t) 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

  9. 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.

  10. 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…

  11. 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.

  12. 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

  13. 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. PMID:26863232

  14. 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.

  15. Detection and Quantification of Magnetically Labeled Cells by Cellular MRI

    PubMed Central

    Liu, Wei; Frank, Joseph A.

    2008-01-01

    Labeling cells with superparamagnetic iron oxide (SPIO) nanoparticles, paramagnetic contrast agent (gadolinium) or perfluorocarbons allows for the possibility of tracking single or clusters of labeled cells within target tissues following either direct implantation or intravenous injection. This review summarizes the practical issues regarding detection and quantification of magnetically labeled cells with various MRI contrast agents with a focus on SPIO nanoparticles. PMID:18995978

  16. 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…

  17. 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. PMID:21845960

  18. SERS Quantification of Entacapone Isomers

    NASA Astrophysics Data System (ADS)

    Marković, Marina; Biljan, Tomislav

    2010-08-01

    Raman spectroscopy, due to its non-destructive character and speed, has found widespread use in pharmaceutical applications [1]. It is also being used for quantifying various isomer mixtures, best known being the quantification of xylene isomers [2-3]. Solid-state isomer quantification of entacapone was earlier reported [4]. Here, we report quantification of isomer mixture of an active pharmaceutical substance, in solution, by SERS.

  19. Selection of reference genes for reverse transcription quantitative real-time PCR normalization in black rockfish (Sebastes schlegeli).

    PubMed

    Liman, Ma; Wenji, Wang; Conghui, Liu; Haiyang, Yu; Zhigang, Wang; Xubo, Wang; Jie, Qi; Quanqi, Zhang

    2013-09-01

    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique widely used for quantification of mRNA transcription. Data normalization is an indispensable process for RT-qPCR and reference genes are most commonly used to normalize RT-qPCR and to reduce possible errors generated in the quantification of genes among several proposed methods. To date, RT-qPCR has been used in terms of gene expression studies in black rockfish (Sebastes schlegeli) but the majority of published RT-qPCR studies still lack proper validation of the reference genes. In the present study, mRNA transcription profiles of eight putative reference genes (18S rRNA, ACTB, GAPDH, TUBA, RPL17, EF1A, HPRT, and B2M) were examined using RT-qPCR in different tissues and larvae developmental stages of black rockfish. Three common statistical algorithms (geNorm, NormFinder, and BestKeeper) were used to assess expression stability and select the most stable genes for gene normalization. Two reference genes, RPL17 and EF1A showed high stability in black rockfish tissue analysis, while GAPDH was the least stable gene. During larvae developmental stages, EF1A, RPL17 and ACTB were identified as the optimal reference genes for data normalization, whereas B2M appeared unsuitable as the reference gene. In summary, our results could provide a useful guideline for reference gene selection and enable more accurate normalization of gene expression data in gene expression studies of black rockfish.

  20. Quantification of gap junction selectivity.

    PubMed

    Ek-Vitorín, Jose F; Burt, Janis M

    2005-12-01

    Gap junctions, which are essential for functional coordination and homeostasis within tissues, permit the direct intercellular exchange of small molecules. The abundance and diversity of this exchange depends on the number and selectivity of the comprising channels and on the transjunctional gradient for and chemical character of the permeant molecules. Limited knowledge of functionally significant permeants and poor detectability of those few that are known have made it difficult to define channel selectivity. Presented herein is a multifaceted approach to the quantification of gap junction selectivity that includes determination of the rate constant for intercellular diffusion of a fluorescent probe (k2-DYE) and junctional conductance (gj) for each junction studied, such that the selective permeability (k2-DYE/gj) for dyes with differing chemical characteristics or junctions with differing connexin (Cx) compositions (or treatment conditions) can be compared. In addition, selective permeability can be correlated using single-channel conductance when this parameter is also measured. Our measurement strategy is capable of detecting 1) rate constants and selective permeabilities that differ across three orders of magnitude and 2) acute changes in that rate constant. Using this strategy, we have shown that 1) the selective permeability of Cx43 junctions to a small cationic dye varied across two orders of magnitude, consistent with the hypothesis that the various channel configurations adopted by Cx43 display different selective permeabilities; and 2) the selective permeability of Cx37 vs. Cx43 junctions was consistently and significantly lower. PMID:16093281

  1. 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

  2. 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.

  3. Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV).

    PubMed

    Duan, X; Nauwynck, H J; Pensaert, M B

    1997-05-01

    Sixteen 6 week old conventional pigs were inoculated by aerosol with a European strain of porcine reproductive and respiratory syndrome virus (PRRSV). Virus replication was followed by virus titration and immunofluorescence in the lungs and in associated and distant lymphoid tissues at 3, 14, 21, 35, 42 and 82 days post-inoculation (DPI). PRRSV replication was detected in alveolar macrophages, lungs, tonsils, spleen, retropharyngeal lymph nodes, bronchial lymph nodes and thoracic aortic lymph nodes at 3 DPI. The same tissues, except retropharyngeal and thoracic aortic lymph nodes, were PRRSV positive at 14 DPI. Lungs and alveolar macrophages were PRRSV positive until 35 DPI. PRRSV was not detected in heart, peripheral blood mononuclear cells and bone marrow cells. Viremia was detected from 3 to 28 DPI. Not more than 2% of alveolar macrophages were PRRSV positive even during the acute stage of infection. 80 to 94% of the PRRSV infected cells in the lungs and in lung lavaged cells were identified as macrophages using a porcine macrophage specific monoclonal antibodies. In the lymph nodes and spleen, 100% of the infected cells were macrophages. Anti-PRRSV antibodies were detected by a blocking ELISA as early as 7 DPI. the antibody titre gradually increased to reach a geometric mean titre (GMT) of 160 at 35 DPI. It remained at that level until the end of the study. These findings clearly demonstrate that PRRSV has a tropism for macrophages. PRRSV mainly replicates in macrophages of the lymphoid tissues and lungs in the acute phase of infection and persists in the lung macrophages.

  4. Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.

    PubMed

    Zhu, Xiaoyang; Li, Xueping; Chen, Weixin; Chen, Jianye; Lu, Wangjin; Chen, Lei; Fu, Danwen

    2012-01-01

    Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.

  5. Automated Quantification of Tumor Viability in a Rabbit Liver Tumor Model after Chemoembolization Using Infrared Imaging.

    PubMed

    D'inca, Hadrien; Namur, Julien; Ghegediban, Saida Homayra; Wassef, Michel; Pascale, Florentina; Laurent, Alexandre; Manfait, Michel

    2015-07-01

    The rabbit VX2 tumor is a fast-growing carcinoma model commonly used to study new therapeutic devices, such as catheter-based therapies for patients with inoperable hepatocellular carcinoma. The evaluation of tumor viability after such locoregional therapies is essential to directing hepatocellular carcinoma management. We used infrared microspectroscopy for the automatic characterization and quantification of the VX2 liver tumor viability after drug-eluting beads transarterial chemoembolization (DEB-TACE). The protocol consisted of K-means clustering followed by principal component analysis (PCA) and linear discriminant analysis (LDA). The K-means clustering was used to classify the spectra from the infrared images of control or treated tumors and to build a database of many tissue spectra. On the basis of this reference library, the PCA-LDA analysis was used to build a predictive model to identify and quantify automatically tumor viability on unknown tissue sections. For the DEB group, the LDA model determined that the surface of tumor necrosis represented 91.6% ± 8.9% (control group: 33.1% ± 19.6%; Mann-Whitney P = 0.0004) and the viable tumor 2.6% ± 4% (control group: 62.2% ± 15.2%; Mann-Whitney P = 0.0004). Tissue quantification measurements correlated well with tumor necrosis (r = 0.827, P < 0.0001) and viable tumor (r = 0.840, P < 0.0001). Infrared imaging and PCA-LDA analysis could be helpful for easily assessing tumor viability. PMID:25979795

  6. Automated Quantification of Tumor Viability in a Rabbit Liver Tumor Model after Chemoembolization Using Infrared Imaging.

    PubMed

    D'inca, Hadrien; Namur, Julien; Ghegediban, Saida Homayra; Wassef, Michel; Pascale, Florentina; Laurent, Alexandre; Manfait, Michel

    2015-07-01

    The rabbit VX2 tumor is a fast-growing carcinoma model commonly used to study new therapeutic devices, such as catheter-based therapies for patients with inoperable hepatocellular carcinoma. The evaluation of tumor viability after such locoregional therapies is essential to directing hepatocellular carcinoma management. We used infrared microspectroscopy for the automatic characterization and quantification of the VX2 liver tumor viability after drug-eluting beads transarterial chemoembolization (DEB-TACE). The protocol consisted of K-means clustering followed by principal component analysis (PCA) and linear discriminant analysis (LDA). The K-means clustering was used to classify the spectra from the infrared images of control or treated tumors and to build a database of many tissue spectra. On the basis of this reference library, the PCA-LDA analysis was used to build a predictive model to identify and quantify automatically tumor viability on unknown tissue sections. For the DEB group, the LDA model determined that the surface of tumor necrosis represented 91.6% ± 8.9% (control group: 33.1% ± 19.6%; Mann-Whitney P = 0.0004) and the viable tumor 2.6% ± 4% (control group: 62.2% ± 15.2%; Mann-Whitney P = 0.0004). Tissue quantification measurements correlated well with tumor necrosis (r = 0.827, P < 0.0001) and viable tumor (r = 0.840, P < 0.0001). Infrared imaging and PCA-LDA analysis could be helpful for easily assessing tumor viability.

  7. 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.

  8. Simple quantification of in planta fungal biomass.

    PubMed

    Ayliffe, Michael; Periyannan, Sambasivam K; Feechan, Angela; Dry, Ian; Schumann, Ulrike; Lagudah, Evans; Pryor, Anthony

    2014-01-01

    An accurate assessment of the disease resistance status of plants to fungal pathogens is an essential requirement for the development of resistant crop plants. Many disease resistance phenotypes are partial rather than obvious immunity and are frequently scored using subjective qualitative estimates of pathogen development or plant disease symptoms. Here we report a method for the accurate comparison of total fungal biomass in plant tissues. This method, called the WAC assay, is based upon the specific binding of the plant lectin wheat germ agglutinin to fungal chitin. The assay is simple, high-throughput, and sensitive enough to discriminate between single Puccinia graminis f.sp tritici infection sites on a wheat leaf segment. It greatly lends itself to replication as large volumes of tissue can be pooled from independent experiments and assayed to provide truly representative quantification, or, alternatively, fungal growth on a single, small leaf segment can be quantified. In addition, as the assay is based upon a microscopic technique, pathogen infection sites can also be examined at high magnification prior to quantification if desired and average infection site areas are determined. Previously, we have demonstrated the application of the WAC assay for quantifying the growth of several different pathogen species in both glasshouse grown material and large-scale field plots. Details of this method are provided within.

  9. Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii

    PubMed Central

    Jiang, Hucheng; Qian, Zhaojun; Lu, Wei; Ding, Huaiyu; Yu, Hongwei; Wang, Hui; Li, Jiale

    2015-01-01

    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. PMID:26370979

  10. 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

  11. A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR.

    PubMed

    Staines, Karen; Batra, Ambalika; Mwangi, William; Maier, Helena J; Van Borm, Steven; Young, John R; Fife, Mark; Butter, Colin

    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

  12. Real-Time PCR for Gene Expression Quantification in Asthma.

    PubMed

    Segundo-Val, Ignacio San; García-Solaesa, Virginia; García-Sánchez, Asunción

    2016-01-01

    The quantitative real-time PCR (qPCR) has become the reference technique for studying gene expression in recent years. The application of qPCR to the study of asthma provides very useful information regarding the gene expression mechanisms. The quantification of RNA from cDNA can be performed by using fluorescent dyes or specific sequence probes. Here, we describe the protocol to quantify gene expression levels using SYBR Green as fluorescent dye. The protocol starts with the RNA extraction, followed by reverse transcription to obtain cDNA, quantification and finally data analysis.

  13. Real-Time PCR for Gene Expression Quantification in Asthma.

    PubMed

    Segundo-Val, Ignacio San; García-Solaesa, Virginia; García-Sánchez, Asunción

    2016-01-01

    The quantitative real-time PCR (qPCR) has become the reference technique for studying gene expression in recent years. The application of qPCR to the study of asthma provides very useful information regarding the gene expression mechanisms. The quantification of RNA from cDNA can be performed by using fluorescent dyes or specific sequence probes. Here, we describe the protocol to quantify gene expression levels using SYBR Green as fluorescent dye. The protocol starts with the RNA extraction, followed by reverse transcription to obtain cDNA, quantification and finally data analysis. PMID:27300530

  14. Quantification of toxicological effects for dichloromethane. Draft report (Final)

    SciTech Connect

    Not Available

    1990-04-01

    The source documents for background information used to develop the report on the quantification of toxicological effects for dichloromethane are the health assessment document (HAD) for dichloromethane and a subsequent addendum to the HAD (U.S. EPA, 1985b). In addition, some references published since 1985 are discussed. To summarize the results of the quantification of toxicological effects, a One-day Health Advisory of 10,000 ug/L for a 10-kg child was calculated, based on an acute oral study in rats reported by Kimura et al. (1971). No suitable data for the derivation of a Ten-day Health Advisory were found in the available literature.

  15. 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

  16. 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.

  17. Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. II. Identification and quantification of 19-norsteroids responsible for 19-norandrosterone and 19-noretiocholanolone excretion in human urine.

    PubMed

    De Wasch, K; Le Bizec, B; De Brabander, H; André, F; Impens, S

    2001-01-01

    In previous work (Le Bizec et al., Rapid Commun. Mass Spectrom. 2000; 14: 1058), it was demonstrated that a boar meal intake could lead to possible false accusations of abuse of 17beta-nortestosterone in antidoping control. The aim of the present study was to identify and quantify endogenous 19-norsteroids in boar edible tissue at concentrations that can alter the steroid urinary profile in humans, and lead to excretion of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). The samples were analysed in two laboratories. The methodologies used for extraction and detection (GC/MS(EI) and LC/MS/MS(APCI+)) are compared and discussed. 19-Norandrostenedione (NAED), 17beta- and 17alpha-nortestosterone (bNT, aNT), and 17beta- and 17alpha-testosterone (bT, aT) were quantified. The largest concentrations of NAED and bNT were observed in testicles (83 and 172 microg/kg), liver (17 and 63 microg/kg) and kidney (45 and 38 microg/kg). A correlation between the bNT and NAED content of a typical meal prepared with boar parts and the excreted concentrations of 19-NA and 19-NE in human urine was demonstrated.

  18. '2' much mitral tissue.

    PubMed

    Barriales-Villa, Roberto; Pérez-Martínez, Fernando; Alvarez-Juárez, Ana I; Vázquez-Rey, Eugenia; Castro-Beiras, Alfonso

    2010-03-01

    A 73-year-old female patient was referred for evaluation after suffering an atypical chest pain. Physical examination and ECG were normal. The echocardiogram showed a normal ventricular function. Attached to the anterior leaflet an accessory mitral valve tissue was identified. In systole, this mitral tissue creates an image that looks like the 'number 2'.

  19. 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

  20. Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq.

    PubMed

    Zhan, Cheng; Zhang, Yongxing; Ma, Jun; Wang, Lin; Jiang, Wei; Shi, Yu; Wang, Qun

    2014-04-01

    Although the accuracy of quantitative real-time polymerase chain reaction (qRT-PCR) is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing (RNA-Seq) to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression profiles of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes (B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC, and YWHAZ), the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes (ACTB) and the 5 additional candidate reference genes (EEF1A1, FAU, RPS9, RPS11, and RPS14) were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEF1A1, FAU, RPS9, RPS11, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context.

  1. 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.

  2. 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

  3. Prediction and Quantification of Splice Events from RNA-Seq Data

    PubMed Central

    Goldstein, Leonard D.; Cao, Yi; Pau, Gregoire; Lawrence, Michael; Wu, Thomas D.; Seshagiri, Somasekar; Gentleman, Robert

    2016-01-01

    Analysis of splice variants from short read RNA-seq data remains a challenging problem. Here we present a novel method for the genome-guided prediction and quantification of splice events from RNA-seq data, which enables the analysis of unannotated and complex splice events. Splice junctions and exons are predicted from reads mapped to a reference genome and are assembled into a genome-wide splice graph. Splice events are identified recursively from the graph and are quantified locally based on reads extending across the start or end of each splice variant. We assess prediction accuracy based on simulated and real RNA-seq data, and illustrate how different read aligners (GSNAP, HISAT2, STAR, TopHat2) affect prediction results. We validate our approach for quantification based on simulated data, and compare local estimates of relative splice variant usage with those from other methods (MISO, Cufflinks) based on simulated and real RNA-seq data. In a proof-of-concept study of splice variants in 16 normal human tissues (Illumina Body Map 2.0) we identify 249 internal exons that belong to known genes but are not related to annotated exons. Using independent RNA samples from 14 matched normal human tissues, we validate 9/9 of these exons by RT-PCR and 216/249 by paired-end RNA-seq (2 x 250 bp). These results indicate that de novo prediction of splice variants remains beneficial even in well-studied systems. An implementation of our method is freely available as an R/Bioconductor package SGSeq. PMID:27218464

  4. Quantification of Glutathione in Caenorhabditis elegans

    PubMed Central

    Caito, Samuel W.; Aschner, Michael

    2015-01-01

    Glutathione (GSH) is the most abundant intracellular thiol with diverse functions from redox signaling, xenobiotic detoxification, and apoptosis. The quantification of GSH is an important measure for redox capacity and oxidative stress. This protocol quantifies total GSH from Caenorhabditis elegans, an emerging model organism for toxicology studies. GSH is measured using the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) cycling method originally created for cell and tissue samples but optimized for whole worm extracts. DTNB reacts with GSH to from a 5′-thio-2-nitrobenzoic acid (TNB) chromophore with maximum absorbance of 412 nm. This method is both rapid and sensitive, making it ideal for studies involving a large number of transgenic nematode strains. PMID:26309452

  5. Quantification of [18F]DPA-714 binding in the human brain: initial studies in healthy controls and Alzheimer's disease patients.

    PubMed

    Golla, Sandeep S V; Boellaard, Ronald; Oikonen, Vesa; Hoffmann, Anja; van Berckel, Bart N M; Windhorst, Albert D; Virta, Jere; Haaparanta-Solin, Merja; Luoto, Pauliina; Savisto, Nina; Solin, Olof; Valencia, Ray; Thiele, Andrea; Eriksson, Jonas; Schuit, Robert C; Lammertsma, Adriaan A; Rinne, Juha O

    2015-05-01

    Fluorine-18 labelled N,N-diethyl-2-(2-[4-(2-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo[1,5-α]pyrimidine-3-yl)acetamide ([(18)F]DPA-714) binds to the 18-kDa translocator protein (TSPO) with high affinity. The aim of this initial methodological study was to develop a plasma input tracer kinetic model for quantification of [(18)F]DPA-714 binding in healthy subjects and Alzheimer's disease (AD) patients, and to provide a preliminary assessment whether there is a disease-related signal. Ten AD patients and six healthy subjects underwent a dynamic positron emission tomography (PET) study along with arterial sampling and a scan protocol of 150 minutes after administration of 250 ± 10 MBq [(18)F]DPA-714. The model that provided the best fits to tissue time activity curves (TACs) was selected based on Akaike Information Criterion and F-test. The reversible two tissue compartment plasma input model with blood volume parameter was the preferred model for quantification of [(18)F]DPA-714 kinetics, irrespective of scan duration, volume of interest, and underlying volume of distribution (VT). Simplified reference tissue model (SRTM)-derived binding potential (BPND) using cerebellar gray matter as reference tissue correlated well with plasma input-based distribution volume ratio (DVR). These data suggest that [(18)F]DPA-714 cannot be used for separating individual AD patients from healthy subjects, but further studies including TSPO binding status are needed to substantiate these findings. PMID:25649991

  6. 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.

  7. Dynamic contrast-enhanced ultrasound for quantification of tissue perfusion.

    PubMed

    Fröhlich, Eckhart; Muller, Reinhold; Cui, Xin-Wu; Schreiber-Dietrich, Dagmar; Dietrich, Christoph F

    2015-02-01

    Dynamic contrast-enhanced ultrasound (US) imaging, a technique that uses microbubble contrast agents with diagnostic US, has recently been technically summarized and reviewed by a European Federation of Societies for Ultrasound in Medicine and Biology position paper. However, the practical applications of this imaging technique were not included. This article reviews and discusses the published literature on the clinical use of dynamic contrast-enhanced US. This review finds that dynamic contrast-enhanced US imaging is the most sensitive cross-sectional real-time method for measuring the perfusion of parenchymatous organs noninvasively. It can measure parenchymal perfusion and therefore can differentiate between benign and malignant tumors. The most important routine clinical role of dynamic contrast-enhanced US is the prediction of tumor responses to chemotherapy within a very short time, shorter than using Response Evaluation Criteria in Solid Tumors criteria. Other applications found include quantifying the hepatic transit time, diabetic kidneys, transplant grafts, and Crohn disease. In addition, the problems involved in using dynamic contrast-enhanced US are discussed.

  8. 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)

  9. 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.

  10. 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. PMID:21744516

  11. 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.

  12. Identification of suitable reference genes in mangrove Aegiceras corniculatum under abiotic stresses.

    PubMed

    Peng, Ya-Lan; Wang, You-Shao; Gu, Ji-Dong

    2015-10-01

    Gene expression studies could provide insight into the physiological mechanisms and strategies used by plants under stress conditions. Selection of suitable internal control gene(s) is essential to accurately assess gene expression levels. For the mangrove plant, Aegiceras corniculatum, reliable reference genes to normalize real-time quantitative PCR data have not been previously investigated. In this study, the expression stabilities of five candidate reference genes [glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 18SrRNA, β-Actin, 60S ribosomal protein L2, and elongation factor-1-A] were determined in leaves of A. corniculatum treated by cold, drought, salt, heavy metals, and pyrene and in different tissues of A. corniculatum under normal condition. Two software programs (geNorm and NormFinder) were employed to analyze and rank the tested genes. Results showed that GAPDH was the most suitable reference gene in A. corniculatum and the combination of two or three genes was recommended for greater accuracy. To assess the value of these tested genes as internal controls, the relative quantifications of CuZnSOD gene were also conducted. Results showed that the relative expression levels of CuZnSOD gene varied depending on the internal reference genes used, which highlights the importance of the choice of suitable internal controls in gene expression studies. Furthermore, the results also confirmed that GAPDH was a suitable reference gene for qPCR normalization in A. corniculatum under abiotic stresses. Identification of A. corniculatum reference gens in a wide range of experimental samples will provide a useful reference in future gene expression studies in this species, particularly involving similar stresses. PMID:25980489

  13. Identification of suitable reference genes in mangrove Aegiceras corniculatum under abiotic stresses.

    PubMed

    Peng, Ya-Lan; Wang, You-Shao; Gu, Ji-Dong

    2015-10-01

    Gene expression studies could provide insight into the physiological mechanisms and strategies used by plants under stress conditions. Selection of suitable internal control gene(s) is essential to accurately assess gene expression levels. For the mangrove plant, Aegiceras corniculatum, reliable reference genes to normalize real-time quantitative PCR data have not been previously investigated. In this study, the expression stabilities of five candidate reference genes [glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 18SrRNA, β-Actin, 60S ribosomal protein L2, and elongation factor-1-A] were determined in leaves of A. corniculatum treated by cold, drought, salt, heavy metals, and pyrene and in different tissues of A. corniculatum under normal condition. Two software programs (geNorm and NormFinder) were employed to analyze and rank the tested genes. Results showed that GAPDH was the most suitable reference gene in A. corniculatum and the combination of two or three genes was recommended for greater accuracy. To assess the value of these tested genes as internal controls, the relative quantifications of CuZnSOD gene were also conducted. Results showed that the relative expression levels of CuZnSOD gene varied depending on the internal reference genes used, which highlights the importance of the choice of suitable internal controls in gene expression studies. Furthermore, the results also confirmed that GAPDH was a suitable reference gene for qPCR normalization in A. corniculatum under abiotic stresses. Identification of A. corniculatum reference gens in a wide range of experimental samples will provide a useful reference in future gene expression studies in this species, particularly involving similar stresses.

  14. 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.

  15. 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. PMID:26874621

  16. 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.

  17. The transcriptome of the reference potato genome Solanum tuberosum Group Phureja clone DM1-3 516R44.

    PubMed

    Massa, Alicia N; Childs, Kevin L; Lin, Haining; Bryan, Glenn J; Giuliano, Giovanni; Buell, C Robin

    2011-01-01

    Advances in molecular breeding in potato have been limited by its complex biological system, which includes vegetative propagation, autotetraploidy, and extreme heterozygosity. The availability of the potato genome and accompanying gene complement with corresponding gene structure, location, and functional annotation are powerful resources for understanding this complex plant and advancing molecular breeding efforts. Here, we report a reference for the potato transcriptome using 32 tissues and growth conditions from the doubled monoploid Solanum tuberosum Group Phureja clone DM1-3 516R44 for which a genome sequence is available. Analysis of greater than 550 million RNA-Seq reads permitted the detection and quantification of expression levels of over 22,000 genes. Hierarchical clustering and principal component analyses captured the biological variability that accounts for gene expression differences among tissues suggesting tissue-specific gene expression, and genes with tissue or condition restricted expression. Using gene co-expression network analysis, we identified 18 gene modules that represent tissue-specific transcriptional networks of major potato organs and developmental stages. This information provides a powerful resource for potato research as well as studies on other members of the Solanaceae family.

  18. 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.…

  19. Pitfalls in target mRNA quantification for real-time quantitative RT-PCR in overload-induced skeletal muscle hypertrophy.

    PubMed

    Chaillou, T; Malgoyre, A; Banzet, S; Chapot, R; Koulmann, N; Pugnière, P; Beaudry, M; Bigard, X; Peinnequin, A

    2011-02-24

    Quantifying target mRNA using real-time quantitative reverse transcription-polymerase chain reaction requires an accurate normalization method. Determination of normalization factors (NFs) based on validated reference genes according to their relative stability is currently the best standard method in most usual situations. This method controls for technical errors, but its physiological relevance requires constant NF values for a fixed weight of tissue. In the functional overload model, the increase in the total RNA concentration must be considered in determining the NF values. Here, we pointed out a limitation of the classical geNorm-derived normalization. geNorm software selected reference genes despite that the NF values extensively varied under experiment. Only the NF values calculated from four intentionally selected genes were constant between groups. However, a normalization based on these genes is questionable. Indeed, three out of four genes belong to the same functional class (negative regulator of muscle mass), and their use is physiological nonsense in a hypertrophic model. Thus, we proposed guidelines for optimizing target mRNA normalization and quantification, useful in models of muscle mass modulation. In our study, the normalization method by multiple reference genes was not appropriate to compare target mRNA levels between overloaded and control muscles. A solution should be to use an absolute quantification of target mRNAs per unit weight of tissue, without any internal normalization. Even if the technical variations will stay present as a part of the intergroup variations, leading to less statistical power, we consider this method acceptable because it will not generate misleading results.

  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. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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…

  6. [Influence of exogenous salicylic acid on the level of phytohormones in tissues of Phlox paniculata and Phlox setacea leaves with special reference to resistance against the powdery mildew causative agent Erysiphe cichoracearum D.C. f. phlogis Jacz].

    PubMed

    Talieva, M N; Kondrat'eva, V V

    2002-01-01

    We studied the effects of exogenous salicylic acid on the level of endogenous cytokinins and abscisic and salicylic acids in the tissues of leaves of phloxes contrasting in resistance against the powdery mildew causative agent: susceptible Phlox paniculata L. and resistant Ph. setacea L. Studies were carried out under the conditions of biotic stress. The initial level of salicylic and abscisic acids and cytokinins is the highest in the resistant phlox species. After treatment with salicylic species, the total level of cytokinins and endogenous salicylic acid increased in both species. When the treated phlox species were infected by the powdery mildew causative agent, the level of abscisic and salicylic acids increased in the susceptible Ph. paniculata, while that of cytokinins increased in the resistant Ph. setacea. The role of salicylic acid in the induction of plant defense reactions against phytopathogens is discussed.

  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. American Indian Reference Book.

    ERIC Educational Resources Information Center

    1976

    Designed to aid librarians, school teachers, and others in need of American Indian references and reference sources, this compilation covers a wide variety of material which has generally been scattered throughout various individual references. Specifically, this reference book includes: (1) Location of Tribes by State; (2) Locations of Tribes by…

  9. Personal Reference in English.

    ERIC Educational Resources Information Center

    Murphy, Gregory L.

    1988-01-01

    Presents a systematic investigation of the factors underlying the choice of personal reference. Five experiments examine how native-English-speaking undergraduates of Brown University refer to a professor or student in various situations. The Rule of Polite Reference (RPR) explains how speakers choose the way they will refer to a person in a given…

  10. 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.

  11. 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;…

  12. Thermomechanical analysis of soft-tissue thermotherapy.

    PubMed

    Aksan, Alptekin; McGrath, John J

    2003-10-01

    Soft-tissue thermotherapy based on sub-ablative heating of collagenous tissues finds wide-spread application in medicine such as tissue welding, thermokeratoplasty, skin resurfacing, elimination of discogenic pain in the spine and treatment of joint instability. In this paper, heat-induced thermomechanical response characteristics of collagenous tissues are quantified by means of in vitro experimentation with a representative model tissue (New Zealand white rabbit patellar tendon). Three distinct heat-induced thermomechanical response regimes (defined by the rate of deformation and the variation of material properties) are identified. Arrhenius damage integral representation of collagenous tissue thermal history is shown to be adequate in establishing the master response curves for quantification of thermomechanical response for modeling purposes. The trade-off between the improved kinematical stability and compromised mechanical stability of the heated collagenous tissue is shown to be the major challenge hindering the success of subablative thermotherapies.

  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). Epithelial tissue ...

  14. Development of magnetic resonance technology for noninvasive boron quantification

    SciTech Connect

    Bradshaw, K.M.

    1990-11-01

    Boron magnetic resonance imaging (MRI) and spectroscopy (MRS) were developed in support of the noninvasive boron quantification task of the Idaho National Engineering Laboratory (INEL) Power Burst Facility/Boron Neutron Capture Therapy (PBF/BNCT) program. The hardware and software described in this report are modifications specific to a GE Signa{trademark} MRI system, release 3.X and are necessary for boron magnetic resonance operation. The technology developed in this task has been applied to obtaining animal pharmacokinetic data of boron compounds (drug time response) and the in-vivo localization of boron in animal tissue noninvasively. 9 refs., 21 figs.

  15. Three-dimensional reconstruction and quantification of cervical carcinoma invasion fronts from histological serial sections.

    PubMed

    Braumann, Ulf-Dietrich; Kuska, Jens-Peer; Einenkel, Jens; Horn, Lars-Christian; Löffler, Markus; Höckel, Michael

    2005-10-01

    The analysis of the three-dimensional (3-D) structure of tumoral invasion fronts of carcinoma of the uterine cervix is the prerequisite for understanding their architectural-functional relationship. The variation range of the invasion patterns known so far reaches from a smooth tumor-host boundary surface to more diffusely spreading patterns, which all are supposed to have a different prognostic relevance. As a very decisive limitation of previous studies, all morphological assessments just could be done verbally referring to single histological sections. Therefore, the intention of this paper is to get an objective quantification of tumor invasion based on 3-D reconstructed tumoral tissue data. The image processing chain introduced here is capable to reconstruct selected parts of tumor invasion fronts from histological serial sections of remarkable extent (90-500 slices). While potentially gaining good accuracy and reasonably high resolution, microtome cutting of large serial sections especially may induce severe artifacts like distortions, folds, fissures or gaps. Starting from stacks of digitized transmitted light color images, an overall of three registration steps are the main parts of the presented algorithm. By this, we achieved the most detailed 3-D reconstruction of the invasion of solid tumors so far. Once reconstructed, the invasion front of the segmented tumor is quantified using discrete compactness.

  16. 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.

  17. 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.

  18. Assessment methods for angiogenesis and current approaches for its quantification

    PubMed Central

    AlMalki, Waleed Hassan; Shahid, Imran; Mehdi, Abeer Yousaf; Hafeez, Muhammad Hassan

    2014-01-01

    Angiogenesis is a physiological process which describes the development of new blood vessels from the existing vessels. It is a common and the most important process in the formation and development of blood vessels, so it is supportive in the healing of wounds and granulation of tissues. The different assays for the evaluation of angiogenesis have been described with distinct advantages and some limitations. In order to develop angiogenic and antiangiogenic techniques, continuous efforts have been resulted to give animal models for more quantitative analysis of angiogenesis. Most of the studies on angiogenic inducers and inhibitors rely on various models, both in vitro, in vivo and in ova, as indicators of efficacy. The angiogenesis assays are very much helpful to test efficacy of both pro- and anti- angiogenic agents. The development of non-invasive procedures for quantification of angiogenesis will facilitate this process significantly. The main objective of this review article is to focus on the novel and existing methods of angiogenesis and their quantification techniques. These findings will be helpful to establish the most convenient methods for the detection, quantification of angiogenesis and to develop a novel, well tolerated and cost effective anti-angiogenic treatment in the near future. PMID:24987169

  19. Quantification of splice variants using molecular beacon or scorpion primers.

    PubMed

    Taveau, Mathieu; Stockholm, Daniel; Spencer, Melissa; Richard, Isabelle

    2002-06-15

    Uncovering the relationship between the generation of alternative transcripts and cellular processes is of crucial importance in the exploration of a gene's biology. The description and quantification of the spatiotemporal splicing pattern can be one method to select the most interesting transcripts for future studies. Fluorescence-based real-time quantitative RT-PCR has recently revolutionized the possibilities for transcriptional quantification studies. In this report, Molecular Beacon and Scorpion probes have been tested as new possibilities for determining the expression level of alternative transcripts. We validated these systems by analyzing alternative splicing of exons 6, 15, and 16 of the calpain 3 gene with tissues containing large variation in the ratio of the different transcripts. We determined conditions that demonstrated that boundary probes are useful tools and good alternatives to boundary primers, when developing a system to quantify specific transcripts. We suggest that the choice of a quantification system should depend in part on the structure and base composition of the gene and may have to be determined experimentally.

  20. 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 ...

  1. 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. PMID:20833124

  2. Quantification of perceived macro-uniformity

    NASA Astrophysics Data System (ADS)

    Lee, Ki-Youn; Bang, Yousun; Choh, Heui-Keun

    2011-01-01

    Macro-uniformity refers to the subjective impression of overall uniformity in the print sample. By the efforts of INCITS W1.1 team, macro-uniformity is categorized into five types of attributes: banding, streaks, mottle, gradients, and moiré patterns, and the ruler samples are generated with perceptual scales. W1.1 macro-uniformity ruler is useful for judging the levels of print defect, but it is not an easy task to reproduce the samples having the same perceptual scales at different times in different places. An objective quantification method is more helpful and convenient for developers to analyze print quality and design printing system components. In this paper, we propose a method for measuring perceived macro-uniformity for a given print using a flat-bed scanner. First, banding, 2D noise, and gradients are separately measured, and they are converted to the perceptual scales based on subjective results of each attribute. The correlation coefficients between the measured values of the attributes and the perceptual scales are 0.92, 0.97, and 0.86, respectively. Another subjective test is performed to find the relationship between the overall macro-uniformity and the three attributes. The weighting factors are obtained by the experimental result, and the final macro-uniformity grade is determined by the weighted sums of each attribute.

  3. 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. PMID:24926893

  4. 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.

  5. Best Reference Sources 2001.

    ERIC Educational Resources Information Center

    Coutts, Brian; McConnell, Tamara

    2002-01-01

    Presents an annotated list of the best reference materials published in 2001. Discusses activity in the reference publishing industry; costs; and lists print materials, Web sites, databases, and CD-ROMs. (LRW)

  6. 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. ...

  7. 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…

  8. Intensity-based quantification of fast retinal blood flow in 3D via high resolution resonant Doppler spectral OCT

    NASA Astrophysics Data System (ADS)

    Michaely, R.; Bachmann, A. H.; Villiger, M. L.; Blatter, C.; Lasser, T.; Leitgeb, R. A.

    2007-07-01

    Resonant Doppler Fourier Domain Optical Coherence Tomography is a functional imaging modality for quantifying fast tissue flow. The method profits from the effect of interference fringe blurring in spectrometer-based FDOCT in the presence of sample motion. If the reference path length is changed in resonance with the Doppler frequency of the sample flow the signals of resting structures will be suppressed whereas the signals of blood flow are enhanced. This allows for an easy extraction of vascularization structure. 3D images of blood vessels at the human optic nerve head are obtained with high axial resolution of 8 μm in air and an imaging speed of 17.400 depth profiles per second. An electro-optic modulator allows controlled reference phase shifting during camera integration. A differential approach is presented for the quantification of fast flows that are un-accessible via standard phase sensitive Doppler analysis. Flow velocity analysis extracts only the axial component which is dependent on the orientation of the vessel with respect to the optical axis. 3D information of the segmented vessel structure is readily used to obtain the flow velocity vectors along the individual vessels and to calculate the true angle-corrected flow speed.

  9. Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida)

    PubMed Central

    2011-01-01

    Background Gene expression studies are a prerequisite for understanding the biological function of genes. Because of its high sensitivity and easy use, quantitative PCR (qPCR) has become the gold standard for gene expression quantification. To normalise qPCR measurements between samples, the most prominent technique is the use of stably expressed endogenous control genes, the so called reference genes. However, recent studies show there is no universal reference gene for all biological questions. Roses are important ornamental plants for which there has been no evaluation of useful reference genes for gene expression studies. Results We used three different algorithms (BestKeeper, geNorm and NormFinder) to validate the expression stability of nine candidate reference genes in different rose tissues from three different genotypes of Rosa hybrida and in leaves treated with various stress factors. The candidate genes comprised the classical "housekeeping genes" (Actin, EF-1α, GAPDH, Tubulin and Ubiquitin), and genes showing stable expression in studies in Arabidopsis (PP2A, SAND, TIP and UBC). The programs identified no single gene that showed stable expression under all of the conditions tested, and the individual rankings of the genes differed between the algorithms. Nevertheless the new candidate genes, specifically, PP2A and UBC, were ranked higher as compared to the other traditional reference genes. In general, Tubulin showed the most variable expression and should be avoided as a reference gene. Conclusions Reference genes evaluated as suitable in experiments with Arabidopsis thaliana were stably expressed in roses under various experimental conditions. In most cases, these genes outperformed conventional reference genes, such as EF1-α and Tubulin. We identified PP2A, SAND and UBC as suitable reference genes, which in different combinations may be used for normalisation in expression analyses via qPCR for different rose tissues and stress treatments. However

  10. 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

  11. 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

  12. Quantification of brain endocannabinoid levels: methods, interpretations and pitfalls

    PubMed Central

    Buczynski, Matthew W; Parsons, Loren H

    2010-01-01

    Endocannabinoids play an important role in a diverse range of neurophysiological processes including neural development, neuroimmune function, synaptic plasticity, pain, reward and affective state. This breadth of influence and evidence for altered endocannabinoid signalling in a variety of neuropathologies has fuelled interest in the accurate quantification of these lipids in brain tissue. Established methods for endocannabinoid quantification primarily employ solvent-based lipid extraction with further sample purification by solid phase extraction. In recent years in vivo microdialysis methods have also been developed for endocannabinoid sampling from the brain interstitial space. However, considerable variability in estimates of endocannabinoid content has led to debate regarding the physiological range of concentrations present in various brain regions. This paper provides a critical review of factors that influence the quantification of brain endocannabinoid content as determined by lipid extraction from bulk tissue and by in vivo microdialysis. A variety of methodological issues are discussed including analytical approaches, endocannabinoid extraction and purification, post-mortem changes in brain endocannabinoid content, cellular reactions to microdialysis probe implantation and caveats related to lipid sampling from the extracellular space. The application of these methods for estimating brain endocannabinoid content and the effects of endocannabinoid clearance inhibition are discussed. The benefits, limitations and pitfalls associated with each approach are emphasized, with an eye toward the appropriate interpretation of data gathered by each method. This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x PMID:20590555

  13. Sensitive quantification of somatic mutations using molecular inversion probes.

    PubMed

    Hirani, Rena; Connolly, Ashley R; Putral, Lisa; Dobrovic, Alexander; Trau, Matt

    2011-11-01

    Somatic mutations in DNA can serve as cancer specific biomarkers and are increasingly being used to direct treatment. However, they can be difficult to detect in tissue biopsies because there is often only a minimal amount of sample and the mutations are often masked by the presence of wild type alleles from nontumor material in the sample. To facilitate the sensitive and specific analysis of DNA mutations in tissues, a multiplex assay capable of detecting nucleotide changes in less than 150 cells was developed. The assay extends the application of molecular inversion probes to enable sensitive discrimination and quantification of nucleotide mutations that are present in less than 0.1% of a cell population. The assay was characterized by detecting selected mutations in the KRAS gene, which has been implicated in up to 25% of all cancers. These mutations were detected in a single multiplex assay by incorporating the rapid flow cytometric readout of multiplexable DNA biosensors.

  14. Unilateral Condylar Hyperplasia: A 3-Dimensional Quantification of Asymmetry

    PubMed Central

    Maal, Thomas J. J.; Bergé, Stefaan J.; Becking, Alfred G.

    2013-01-01

    Purpose Objective quantifications of facial asymmetry in patients with Unilateral Condylar Hyperplasia (UCH) have not yet been described in literature. The aim of this study was to objectively quantify soft-tissue asymmetry in patients with UCH and to compare the findings with a control group using a new method. Material and Methods Thirty 3D photographs of patients diagnosed with UCH were compared with 30 3D photographs of healthy controls. As UCH presents particularly in the mandible, a new method was used to isolate the lower part of the face to evaluate asymmetry of this part separately. The new method was validated by two observers using 3D photographs of five patients and five controls. Results A significant difference (0.79 mm) between patients and controls whole face asymmetry was found. Intra- and inter-observer differences of 0.011 mm (−0.034–0.011) and 0.017 mm (−0.007–0.042) respectively were found. These differences are irrelevant in clinical practice. Conclusion After objective quantification, a significant difference was identified in soft-tissue asymmetry between patients with UCH and controls. The method used to isolate mandibular asymmetry was found to be valid and a suitable tool to evaluate facial asymmetry. PMID:23544063

  15. Tissue Microarrays.

    PubMed

    Dancau, Ana-Maria; Simon, Ronald; Mirlacher, Martina; Sauter, Guido

    2016-01-01

    Modern next-generation sequencing and microarray technologies allow for the simultaneous analysis of all human genes on the DNA, RNA, miRNA, and methylation RNA level. Studies using such techniques have lead to the identification of hundreds of genes with a potential role in cancer or other diseases. The validation of all of these candidate genes requires in situ analysis of high numbers of clinical tissues samples. The tissue microarray technology greatly facilitates such analysis. In this method minute tissue samples (typically 0.6 mm in diameter) from up to 1000 different tissues can be analyzed on one microscope glass slide. All in situ methods suitable for histological studies can be applied to TMAs without major changes of protocols, including immunohistochemistry, fluorescence in situ hybridization, or RNA in situ hybridization. Because all tissues are analyzed simultaneously with the same batch of reagents, TMA studies provide an unprecedented degree of standardization, speed, and cost efficiency.

  16. Tissue Tregs.

    PubMed

    Panduro, Marisella; Benoist, Christophe; Mathis, Diane

    2016-05-20

    The immune system is responsible for defending an organism against the myriad of microbial invaders it constantly confronts. It has become increasingly clear that the immune system has a second major function: the maintenance of organismal homeostasis. Foxp3(+)CD4(+) regulatory T cells (Tregs) are important contributors to both of these critical activities, defense being the primary purview of Tregs circulating through lymphoid organs, and homeostasis ensured mainly by their counterparts residing in parenchymal tissues. This review focuses on so-called tissue Tregs. We first survey existing information on the phenotype, function, sustaining factors, and human equivalents of the three best-characterized tissue-Treg populations-those operating in visceral adipose tissue, skeletal muscle, and the colonic lamina propria. We then attempt to distill general principles from this body of work-as concerns the provenance, local adaptation, molecular sustenance, and targets of action of tissue Tregs, in particular.

  17. Validation of reference genes for gene expression studies in Aphis glycines (Hemiptera: Aphididae).

    PubMed

    Bansal, Raman; Mamidala, Praveen; Mian, M A Rouf; Mittapalli, Omprakash; Michel, Andy P

    2012-08-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is a common and robust tool for accurate quantification of mRNA transcripts. To normalize results, a housekeeping gene ([HKG], reference gene or endogenous control gene) is mandatory. Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a significant soybean, Glycine max (L.) Merr., pest, yet gene expression and functional genomics studies are hindered by a lack of stable HKGs. We evaluated seven potential HKGs (SDFS, succinate dehydrogenase flavoprotein subunit; EF1a, elongation factor-la; HEL, helicase; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; RPS9, ribosomal protein S9; TBP, TATA-box binding protein; and UBQ, ubiquitin-conjugating protein) to determine the most efficient HKGs that have stable expression among tissues, developmental stages, and aphids fed on susceptible and host plant-resistant soybean. HKG stability was determined using GeNorm and NormFinder. Results from three different experimental conditions revealed high stability of TBP compared with the other HKGs profiled across the samples assayed. RPS9 showed stable expression among aphids on susceptible and resistant plants, whereas EF1a showed stable expression in tissues and developmental stages. Therefore, we recommend the TBP as a suitable HKG for efficient normalization among treatments, tissues, and developmental stages of A. glycines. In addition, RPS9 may be used for host-plant resistance experiments and EF1a could be considered for testing differential expression across tissues or developmental stages. These results will enable a more accurate and reliable normalization of qRT-PCR data in A. glycines. PMID:22928326

  18. 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. PMID:24036657

  19. 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.

  20. Fast and Sensitive Method for Determination of Domoic Acid in Mussel Tissue.

    PubMed

    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

  1. 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.

  2. 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.

  3. The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale

    PubMed Central

    2010-01-01

    Background Quantitative real-time PCR (qPCR) has been the method of choice for the quantification of mRNA. Due to the various artifactual factors that may affect the accuracy of qPCR, internal reference genes are most often used to normalize qPCR data. Recently, many studies have employed computer programs such as GeNorm, BestKeeper and NormFinder in selecting reference genes, but very few statistically validate the outcomes of these programs. Thus, in this study, we selected reference genes for qPCR of liver and ovary samples of yellow (juvenile), migratory (silver) and 11-KT treated juveniles of New Zealand shortfinned eels (Anguilla australis) using the three computer programs and validate the selected genes statistically using REST 2009 software and the Mann-Whitney test. We also tested for the repeatability of use for the best reference genes by applying them to a data set obtained in a similar experiment conducted the previous year. Results Out of six candidate genes, the combination of 18 s and eef1 was found to be the best statistically validated reference for liver, while in ovary it was l36. However, discrepancies in gene rankings were found between the different programs. Also, statistical validation procedures showed that several genes put forward as being the best by the programs were in fact, regulated, making them unsuitable as reference genes. Additionally, eef1 which was found to be a suitable - though not the top ranked - reference gene for liver tissues in one year, was regulated in another. Conclusions Our study highlights the need for external validations of reference gene selections made by computer programs. Researchers need to be vigilant in validating and reporting the rationale for the use of reference gene in published studies. PMID:20860839

  4. Optical voltage reference

    DOEpatents

    Rankin, R.; Kotter, D.

    1994-04-26

    An optical voltage reference for providing an alternative to a battery source is described. The optical reference apparatus provides a temperature stable, high precision, isolated voltage reference through the use of optical isolation techniques to eliminate current and impedance coupling errors. Pulse rate frequency modulation is employed to eliminate errors in the optical transmission link while phase-lock feedback is employed to stabilize the frequency to voltage transfer function. 2 figures.

  5. 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

  6. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Gentiana macrophylla.

    PubMed

    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 ΔC t, 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

  7. 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

  8. Enhanced characterization of calcified areas in intravascular ultrasound virtual histology images by quantification of the acoustic shadow: validation against computed tomography coronary angiography.

    PubMed

    Broersen, Alexander; de Graaf, Michiel A; Eggermont, Jeroen; Wolterbeek, Ron; Kitslaar, Pieter H; Dijkstra, Jouke; Bax, Jeroen J; Reiber, Johan H C; Scholte, Arthur J

    2016-04-01

    We enhance intravascular ultrasound virtual histology (VH) tissue characterization by fully automatic quantification of the acoustic shadow behind calcified plaque. VH is unable to characterize atherosclerosis located behind calcifications. In this study, the quantified acoustic shadows are considered calcified to approximate the real dense calcium (DC) plaque volume. In total, 57 patients with 108 coronary lesions were included. A novel post-processing step is applied on the VH images to quantify the acoustic shadow and enhance the VH results. The VH and enhanced VH results are compared to quantitative computed tomography angiography (QTA) plaque characterization as reference standard. The correlation of the plaque types between enhanced VH and QTA differs significantly from the correlation with unenhanced VH. For DC, the correlation improved from 0.733 to 0.818. Instead of an underestimation of DC in VH with a bias of 8.5 mm(3), there was a smaller overestimation of 1.1 mm(3) in the enhanced VH. Although tissue characterization within the acoustic shadow in VH is difficult, the novel algorithm improved the DC tissue characterization. This algorithm contributes to accurate assessment of calcium on VH and could be applied in clinical studies.

  9. Enhanced characterization of calcified areas in intravascular ultrasound virtual histology images by quantification of the acoustic shadow: validation against computed tomography coronary angiography.

    PubMed

    Broersen, Alexander; de Graaf, Michiel A; Eggermont, Jeroen; Wolterbeek, Ron; Kitslaar, Pieter H; Dijkstra, Jouke; Bax, Jeroen J; Reiber, Johan H C; Scholte, Arthur J

    2016-04-01

    We enhance intravascular ultrasound virtual histology (VH) tissue characterization by fully automatic quantification of the acoustic shadow behind calcified plaque. VH is unable to characterize atherosclerosis located behind calcifications. In this study, the quantified acoustic shadows are considered calcified to approximate the real dense calcium (DC) plaque volume. In total, 57 patients with 108 coronary lesions were included. A novel post-processing step is applied on the VH images to quantify the acoustic shadow and enhance the VH results. The VH and enhanced VH results are compared to quantitative computed tomography angiography (QTA) plaque characterization as reference standard. The correlation of the plaque types between enhanced VH and QTA differs significantly from the correlation with unenhanced VH. For DC, the correlation improved from 0.733 to 0.818. Instead of an underestimation of DC in VH with a bias of 8.5 mm(3), there was a smaller overestimation of 1.1 mm(3) in the enhanced VH. Although tissue characterization within the acoustic shadow in VH is difficult, the novel algorithm improved the DC tissue characterization. This algorithm contributes to accurate assessment of calcium on VH and could be applied in clinical studies. PMID:26667446

  10. In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.

    PubMed

    Asara, John M; Zhang, Xiang; Zheng, Bin; Christofk, Heather H; Wu, Ning; Cantley, Lewis C

    2006-01-01

    Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.

  11. 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…

  12. Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L.) Dunal.

    PubMed

    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.

  13. 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

  14. Identification and Validation of Reference Genes for Gene Expression Analysis Using Quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)

    PubMed Central

    Gao, Xiwu; Kang, Tinghao; Zhan, Sha; Wan, Hu; Li, Jianhong

    2013-01-01

    Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms. PMID:23874494

  15. 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. PMID:27672374

  16. 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

  17. 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…

  18. 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)

  19. 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…

  20. 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.

  1. Ethics and Reference Services.

    ERIC Educational Resources Information Center

    Danielson, Elena S.

    1997-01-01

    While revised ethical codes provide helpful guidelines, reference archivists face many ethical questions raised by rapidly evolving technology, changing expectations, and inconsistent privacy laws that have no clear answers. Discusses issues related to reference searching, codification of ethics, cultural property and the responsibility of…

  2. 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…

  3. Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk.

    PubMed

    Modesto, P; Peletto, S; Pisoni, G; Cremonesi, P; Castiglioni, B; Colussi, S; Caramelli, M; Bronzo, V; Moroni, P; Acutis, P L

    2013-01-01

    Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal

  4. Integrative analysis of 111 reference human epigenomes

    PubMed Central

    Kundaje, Anshul; Meuleman, Wouter; Ernst, Jason; Bilenky, Misha; Yen, Angela; Kheradpour, Pouya; Zhang, Zhizhuo; Heravi-Moussavi, Alireza; Liu, Yaping; Amin, Viren; Ziller, Michael J; Whitaker, John W; Schultz, Matthew D; Sandstrom, Richard S; Eaton, Matthew L; Wu, Yi-Chieh; Wang, Jianrong; Ward, Lucas D; Sarkar, Abhishek; Quon, Gerald; Pfenning, Andreas; Wang, Xinchen; Claussnitzer, Melina; 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; Mercer, Tim R; Neph, Shane J; Onuchic, Vitor; Polak, Paz; Rajagopal, Nisha; Ray, Pradipta; Sallari, Richard C; Siebenthall, Kyle T; Sinnott-Armstrong, Nicholas; Stevens, Michael; Thurman, Robert E; Wu, Jie; Zhang, Bo; Zhou, Xin; Beaudet, Arthur E; Boyer, Laurie A; De Jager, Philip; Farnham, Peggy J; Fisher, Susan J; Haussler, David; Jones, Steven; Li, Wei; Marra, Marco; McManus, Michael T; Sunyaev, Shamil; Thomson, James A; Tlsty, Thea D; Tsai, Li-Huei; Wang, Wei; Waterland, Robert A; Zhang, Michael; 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

    2015-01-01

    The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but a similar reference has lacked for epigenomic studies. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection to-date of human epigenomes for primary cells and tissues. Here, we describe the integrative analysis of 111 reference human epigenomes generated as part of the program, 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. PMID:25693563

  5. 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.

  6. 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. PMID:25693563

  7. Automated core-penumbra quantification in neonatal ischemic brain injury.

    PubMed

    Ghosh, Nirmalya; Yuan, Xiangpeng; Turenius, Christine I; Tone, Beatriz; Ambadipudi, Kamalakar; Snyder, Evan Y; Obenaus, Andre; Ashwal, Stephen

    2012-12-01

    Neonatal hypoxic-ischemic brain injury (HII) and arterial ischemic stroke (AIS) result in irreversibly injured (core) and salvageable (penumbral) tissue regions. Identification and reliable quantification of salvageable tissue is pivotal to any effective and safe intervention. Magnetic resonance imaging (MRI) is the current standard to distinguish core from penumbra using diffusion-perfusion mismatch (DPM). However, subtle MR signal variations between core-penumbral regions make their visual delineation difficult. We hypothesized that computational analysis of MRI data provides a more accurate assessment of core and penumbral tissue evolution in HII/AIS. We used two neonatal rat-pup models of HII/AIS (unilateral and global hypoxic-ischemia) and clinical data sets from neonates with AIS to test our noninvasive, automated computational approach, Hierarchical Region Splitting (HRS), to detect and quantify ischemic core-penumbra using only a single MRI modality (T2- or diffusion-weighted imaging, T2WI/DWI). We also validated our approach by comparing core-penumbral images (from HRS) to DPM with immunohistochemical validation of HII tissues. Our translational and clinical data results showed that HRS could accurately and reliably distinguish the ischemic core from penumbra and their spatiotemporal evolution, which may aid in the vetting and execution of effective therapeutic interventions as well as patient selection.

  8. Direct visualization and quantification of bone growth into porous titanium implants using micro computed tomography.

    PubMed

    Baril, E; Lefebvre, L P; Hacking, S A

    2011-05-01

    The utility of porous metals for the integration of orthopaedic implants with host bone has been well established. Quantification of the tissue response to cementless implants is laborious and time consuming process requiring tissue processing, embedding, sectioning, polishing, imaging and image analysis. Micro-computed tomography (μCT) is a promising three dimensional (3D) imaging technique to quantify the tissue response to porous metals. However, the suitability and effectiveness of μCT for the quantification of bone ingrowth remains unknown. The purpose of this study was to evaluate and compare bone growth within porous titanium implants using both μCT and traditional hard-tissue histology techniques. Cylindrical implants were implanted in the distal femora and proximal tibiae of a rabbit. After 6 weeks, bone ingrowth was quantified and compared by μCT, light microscopy and backscattered electron microscopy. Quantification of bone volume and implant porosity as determined by μCT compared well with data obtained by traditional histology techniques. Analysis of the 3D dataset showed that bone was present in the pores connected with openings larger 9.4 μm. For pore openings greater than 28.2 μm, the size of the interconnection had little impact on the bone density within the porosity for the titanium foams. PMID:21512898

  9. 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.

  10. Quantification and Localization of Mast Cells in Periapical Lesions

    PubMed Central

    Mahita, VN; Manjunatha, BS; Shah, R; Astekar, M; Purohit, S; Kovvuru, S

    2015-01-01

    Background: Periapical lesions occur in response to chronic irritation in periapical tissue, generally resulting from an infected root canal. Specific etiological agents of induction, participating cell population and growth factors associated with maintenance and resolution of periapical lesions are incompletely understood. Among the cells found in periapical lesions, mast cells have been implicated in the inflammatory mechanism. Aim: Quantifications and the possible role played by mast cells in the periapical granuloma and radicular cyst. Hence, this study is to emphasize the presence (localization) and quantification of mast cells in periapical granuloma and radicular cyst. Materials and Methods: A total of 30 cases and out of which 15 of periapical granuloma and 15 radicular cyst, each along with the case details from the previously diagnosed cases in the department of oral pathology were selected for the study. The gender distribution showed male 8 (53.3%) and females 7 (46.7%) in periapical granuloma cases and male 10 (66.7%) and females 5 (33.3%) in radicular cyst cases. The statistical analysis used was unpaired t-test. Results: Mean mast cell count in periapical granuloma subepithelial and deeper connective tissue, was 12.40 (0.99%) and 7.13 (0.83%), respectively. The mean mast cell counts in subepithelial and deeper connective tissue of radicular cyst were 17.64 (1.59%) and 12.06 (1.33%) respectively, which was statistically significant. No statistical significant difference was noted among males and females. Conclusion: Mast cells were more in number in radicular cyst. Based on the concept that mast cells play a critical role in the induction of inflammation, it is logical to use therapeutic agents to alter mast cell function and secretion, to thwart inflammation at its earliest phases. These findings may suggest the possible role of mast cells in the pathogenesis of periapical lesions. PMID:25861530

  11. 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.

  12. Influence of absorption and scattering on the quantification of fluorescence diffuse optical tomography using normalized data.

    PubMed

    Abascal, Juan Felipe Perez-Juste; Aguirre, Juan; Chamorro-Servent, Judit; Schweiger, Martin; Arridge, Simon; Ripoll, Jorge; Vaquero, Juan J; Desco, Manuel

    2012-03-01

    Reconstruction algorithms for imaging fluorescence in near infrared ranges usually normalize fluorescence light with respect to excitation light. Using this approach, we investigated the influence of absorption and scattering heterogeneities on quantification accuracy when assuming a homogeneous model and explored possible reconstruction improvements by using a heterogeneous model. To do so, we created several computer-simulated phantoms: a homogeneous slab phantom (P1), slab phantoms including a region with a two- to six-fold increase in scattering (P2) and in absorption (P3), and an atlas-based mouse phantom that modeled different liver and lung scattering (P4). For P1, reconstruction with the wrong optical properties yielded quantification errors that increased almost linearly with the scattering coefficient while they were mostly negligible regarding the absorption coefficient. This observation agreed with the theoretical results. Taking the quantification of a homogeneous phantom as a reference, relative quantification errors obtained when wrongly assuming homogeneous media were in the range +41 to +94% (P2), 0.1 to -7% (P3), and -39 to +44% (P4). Using a heterogeneous model, the overall error ranged from -7 to 7%. In conclusion, this work demonstrates that assuming homogeneous media leads to noticeable quantification errors that can be improved by adopting heterogeneous models.

  13. Influence of absorption and scattering on the quantification of fluorescence diffuse optical tomography using normalized data

    NASA Astrophysics Data System (ADS)

    Abascal, Juan Felipe Perez-Juste; Aguirre, Juan; Chamorro-Servent, Judit; Schweiger, Martin; Arridge, Simon; Ripoll, Jorge; Vaquero, Juan J.; Desco, Manuel

    2012-03-01

    Reconstruction algorithms for imaging fluorescence in near infrared ranges usually normalize fluorescence light with respect to excitation light. Using this approach, we investigated the influence of absorption and scattering heterogeneities on quantification accuracy when assuming a homogeneous model and explored possible reconstruction improvements by using a heterogeneous model. To do so, we created several computer-simulated phantoms: a homogeneous slab phantom (P1), slab phantoms including a region with a two- to six-fold increase in scattering (P2) and in absorption (P3), and an atlas-based mouse phantom that modeled different liver and lung scattering (P4). For P1, reconstruction with the wrong optical properties yielded quantification errors that increased almost linearly with the scattering coefficient while they were mostly negligible regarding the absorption coefficient. This observation agreed with the theoretical results. Taking the quantification of a homogeneous phantom as a reference, relative quantification errors obtained when wrongly assuming homogeneous media were in the range +41 to +94% (P2), 0.1 to -7% (P3), and -39 to +44% (P4). Using a heterogeneous model, the overall error ranged from -7 to 7%. In conclusion, this work demonstrates that assuming homogeneous media leads to noticeable quantification errors that can be improved by adopting heterogeneous models.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Genetics Home Reference: sitosterolemia

    MedlinePlus

    ... Sitosterolemia is a condition in which fatty substances (lipids) from vegetable oils, nuts, and other plant-based foods accumulate in the blood and tissues. These lipids are called plant sterols (or phytosterols). Sitosterol is ...

  19. 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...

  20. Value of Information References

    DOE Data Explorer

    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.

  1. A flexible numerical approach for quantification of epistemic uncertainty

    NASA Astrophysics Data System (ADS)

    Chen, Xiaoxiao; Park, Eun-Jae; Xiu, Dongbin

    2013-05-01

    In the field of uncertainty quantification (UQ), epistemic uncertainty often refers to the kind of uncertainty whose complete probabilistic description is not available, largely due to our lack of knowledge about the uncertainty. Quantification of the impacts of epistemic uncertainty is naturally difficult, because most of the existing stochastic tools rely on the specification of the probability distributions and thus do not readily apply to epistemic uncertainty. And there have been few studies and methods to deal with epistemic uncertainty. A recent work can be found in [J. Jakeman, M. Eldred, D. Xiu, Numerical approach for quantification of epistemic uncertainty, J. Comput. Phys. 229 (2010) 4648-4663], where a framework for numerical treatment of epistemic uncertainty was proposed. The method is based on solving an encapsulation problem, without using any probability information, in a hypercube that encapsulates the unknown epistemic probability space. If more probabilistic information about the epistemic variables is known a posteriori, the solution statistics can then be evaluated at post-process steps. In this paper, we present a new method, similar to that of Jakeman et al. but significantly extending its capabilities. Most notably, the new method (1) does not require the encapsulation problem to be in a bounded domain such as a hypercube; (2) does not require the solution of the encapsulation problem to converge point-wise. In the current formulation, the encapsulation problem could reside in an unbounded domain, and more importantly, its numerical approximation could be sought in Lp norm. These features thus make the new approach more flexible and amicable to practical implementation. Both the mathematical framework and numerical analysis are presented to demonstrate the effectiveness of the new approach.

  2. A flexible numerical approach for quantification of epistemic uncertainty

    SciTech Connect

    Chen, Xiaoxiao; Park, Eun-Jae; Xiu, Dongbin

    2013-05-01

    In the field of uncertainty quantification (UQ), epistemic uncertainty often refers to the kind of uncertainty whose complete probabilistic description is not available, largely due to our lack of knowledge about the uncertainty. Quantification of the impacts of epistemic uncertainty is naturally difficult, because most of the existing stochastic tools rely on the specification of the probability distributions and thus do not readily apply to epistemic uncertainty. And there have been few studies and methods to deal with epistemic uncertainty. A recent work can be found in [J. Jakeman, M. Eldred, D. Xiu, Numerical approach for quantification of epistemic uncertainty, J. Comput. Phys. 229 (2010) 4648–4663], where a framework for numerical treatment of epistemic uncertainty was proposed. The method is based on solving an encapsulation problem, without using any probability information, in a hypercube that encapsulates the unknown epistemic probability space. If more probabilistic information about the epistemic variables is known a posteriori, the solution statistics can then be evaluated at post-process steps. In this paper, we present a new method, similar to that of Jakeman et al. but significantly extending its capabilities. Most notably, the new method (1) does not require the encapsulation problem to be in a bounded domain such as a hypercube; (2) does not require the solution of the encapsulation problem to converge point-wise. In the current formulation, the encapsulation problem could reside in an unbounded domain, and more importantly, its numerical approximation could be sought in L{sup p} norm. These features thus make the new approach more flexible and amicable to practical implementation. Both the mathematical framework and numerical analysis are presented to demonstrate the effectiveness of the new approach.