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Sample records for reference tissue quantification

  1. 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

  2. The impact of reference gene selection in quantification of gene expression levels in guinea pig cervical tissues and cells

    PubMed Central

    2013-01-01

    Background Accurate measurements of mRNA expression levels in tissues or cells are crucially dependent on the use of relevant reference genes for normalization of data. In this study we used quantitative real-time PCR and two Excel-based applets (geNorm and BestKeeper) to determine the best reference genes for quantification of target gene mRNA in a complex tissue organ such as the guinea pig cervix. Results Gene expression studies were conducted in cervical epithelium and stroma during pregnancy and parturition and in cultures of primary cells from this tissue. Among 15 reference gene candidates examined, both geNorm and BestKeeper found CLF1 and CLTC to be the most stable in cervical stroma and cervical epithelium, ACTB and PPIB in primary stroma cells, and CLTC and PPIB in primary epithelial cells. The order of stability among the remaining candidate genes was not in such an agreement. Commonly used reference such as GAPDH and B2M demonstrated lower stability. Determination of pairwise variation values for reference gene combinations using geNorm revealed that the geometric mean of the two most stable genes provides sufficient normalization in most cases. However, for cervical stroma tissue in which many reference gene candidates displayed low stability, inclusion of three reference genes in the geometric mean may improve accuracy of target gene expression level analyses. Using the top ranked reference genes we examined the expression levels of target gene PTGS2 in cervical tissue and cultured cervical cells. We compared the results with PTGS2 expression normalized to the least stable gene and found significant differences in gene expression, up to 10-fold in some samples, emphasizing the importance of appropriately selecting reference genes. Conclusions We recommend using the geometric mean of CFL1 and CLTC for normalization of qPCR studies in guinea pig cervical tissue studies, ACTB and PPIB in primary stroma cells and CLTC and PPIB in primary epithelial cells

  3. [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

  4. 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

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

    NASA Astrophysics Data System (ADS)

    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 [11C]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. PMID:27073214

  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. The Simplified Reference Tissue Model with 18F-fallypride PET: Choice of Reference Region

    PubMed Central

    Ishibashi, Kenji; Robertson, Chelsea L.; Mandelkern, Mark A.; Morgan, Andrew T.; London, Edythe D.

    2014-01-01

    The development of high-affinity radiotracers for positron emission tomography has allowed for quantification of dopamine receptors in extrastriatal as well as striatal regions of brain. As these new radiotracers have distinctly different kinetic properties than their predecessors, it is important to examine the suitability of kinetic models to represent their uptake, distribution and in vivo washout. Using the simplified reference tissue model, we investigated the influence of reference region choice on striatal binding potential (BPND) of 18F-fallypride, a high-affinity dopamine D2/D3 receptor ligand. We compared visual cortex and a white matter region (superior longitudinal fasciculus) to the cerebellum, a commonly used reference tissue, in a PET-fallypride study of healthy and methamphetamine-dependent subjects. Compared to cerebellum, use of visual cortex produced significantly greater sample variance in BPND. Use of the white matter region was associated with BPND values and sample variance similar to those obtained with cerebellum, and a larger effect size for the group differences in striatal BPND between healthy and methamphetamine-dependent subjects. Our results do not support the use of visual cortex as a reference region in 18F-fallypride studies, and suggest that white matter may be a reasonable alternative to cerebellum as it displays similar statistical and kinetic properties. PMID:24447617

  13. Segmentation and quantification of adipose tissue by magnetic resonance imaging.

    PubMed

    Hu, Houchun Harry; Chen, Jun; Shen, Wei

    2016-04-01

    In this brief review, introductory concepts in animal and human adipose tissue segmentation using proton magnetic resonance imaging (MRI) and computed tomography are summarized in the context of obesity research. Adipose tissue segmentation and quantification using spin relaxation-based (e.g., T1-weighted, T2-weighted), relaxometry-based (e.g., T1-, T2-, T2*-mapping), chemical-shift selective, and chemical-shift encoded water-fat MRI pulse sequences are briefly discussed. The continuing interest to classify subcutaneous and visceral adipose tissue depots into smaller sub-depot compartments is mentioned. The use of a single slice, a stack of slices across a limited anatomical region, or a whole body protocol is considered. Common image post-processing steps and emerging atlas-based automated segmentation techniques are noted. Finally, the article identifies some directions of future research, including a discussion on the growing topic of brown adipose tissue and related segmentation considerations. PMID:26336839

  14. 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

  15. 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

  16. 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.

  17. Unification of gene expression data applying standard mRNA quantification references for comparable analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High throughput quantitative measurements of gene expression data have problems of reproducibility and comparability due to a lack of standard mRNA quantification references. Efforts have been made to safeguard data fidelity, yet generating quality expression data of inherent value remains a challe...

  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. Development of automated quantification of visceral and subcutaneous adipose tissue volumes from abdominal CT scans

    NASA Astrophysics Data System (ADS)

    Mensink, Sanne D.; Spliethoff, Jarich W.; Belder, Ruben; Klaase, Joost M.; Bezooijen, Roland; Slump, Cornelis H.

    2011-03-01

    This contribution describes a novel algorithm for the automated quantification of visceral and subcutaneous adipose tissue volumes from abdominal CT scans of patients referred for colorectal resection. Visceral and subcutaneous adipose tissue volumes can accurately be measured with errors of 1.2 and 0.5%, respectively. Also the reproducibility of CT measurements is good; a disadvantage is the amount of radiation. In this study the diagnostic CT scans in the work - up of (colorectal) cancer were used. This implied no extra radiation. For the purpose of segmentation alone, a low dose protocol can be applied. Obesity is a well known risk factor for complications in and after surgery. Body Mass Index (BMI) is a widely accepted indicator of obesity, but it is not specific for risk assessment of colorectal surgery. We report on an automated method to quantify visceral and subcutaneous adipose tissue volumes as a basic step in a clinical research project concerning preoperative risk assessment. The outcomes are to be correlated with the surgery results. The hypothesis is that the balance between visceral and subcutaneous adipose tissue together with the presence of calcifications in the major bloodvessels, is a predictive indicator for post - operatieve complications such as anastomotic leak. We start with four different computer simulated humanoid abdominal volumes with tissue values in the appropriate Hounsfield range at different dose levels. With satisfactory numerical results for this test, we have applied the algorithm on over a 100 patient scans and have compared results with manual segmentations by an expert for a smaller pilot group. The results are within a 5% difference. Compared to other studies reported in the literature, reliable values are obtained for visceral and subcutaneous adipose tissue areas.

  1. 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

  2. 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

  3. Semi-parametric time-domain quantification of HR-MAS data from prostate tissue

    PubMed Central

    Ratiney, Helene; Albers, Mark J.; Rabeson, Herald; Kurhanewicz, John

    2011-01-01

    High Resolution – Magic Angle Spinning (HR-MAS) spectroscopy provides rich biochemical profiles that require accurate quantification to permit biomarker identification and to understand the underlying pathological mechanisms. Meanwhile, quantification of HR-MAS data from prostate tissue samples is challenging due to significant overlap between the resonant peaks, the presence of short T2∗ metabolites such as citrate or polyamines (T2 from 25 to 100 msec) and macromolecules, and variations in chemical shifts and T2∗s within a metabolite’s spin systems. Since existing methods do not address these challenges completely, a new quantification method was developed and optimized for HR-MAS data acquired with an ultra short TE and over 30,000 data points. The proposed method, named HR-QUEST (High Resolution – QUEST), iteratively employs the QUEST time-domain semi-parametric strategy with a new model function that incorporates prior knowledge from whole and subdivided metabolite signals. With these features, HR-QUEST is able to independently fit the chemical shifts and T2∗s of a metabolite’s spin systems, a necessity for HR-MAS data. By using the iterative fitting approach, it is able to account for significant contributions from macromolecules and to handle shorter T2 metabolites, such as citrate and polyamines. After subdividing the necessary metabolite basis signals, the root mean square (RMS) of the residual was reduced by 52% for measured HR-MAS data from prostate tissue. Monte Carlo studies on simulated spectra with varied macromolecular contributions showed that the iterative fitting approach (6 iterations) coupled with inclusion of long T2 macromolecule components in the basis set improve the quality of the fit, as assessed by the reduction of the RMS of the residual and of the RMS error of the metabolite signal estimate, by 27% and 71% respectively. With this optimized configuration, HR-QUEST was applied to measured HR-MAS prostate data and reliably

  4. 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

  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. 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.

  7. Quantification of petroleum-type hydrocarbons in avian tissue.

    PubMed

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

    1980-01-01

    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. PMID:7358812

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

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

    PubMed Central

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

    2015-01-01

    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

  16. Identification and quantification of selected chemicals in laser pyrolysis products of mammalian tissues

    NASA Astrophysics Data System (ADS)

    Spleiss, Martin; Weber, Lothar W.; Meier, Thomas H.; Treffler, Bernd

    1995-01-01

    Liver and muscle tissue have been irradiated with a surgical CO2-laser. The prefiltered fumes were adsorbed on different sorbents (activated charcoal type NIOSH and Carbotrap) and desorbed with different solvents (carbondisulphide and acetone). Analysis was done by gas chromatography/mass spectrometry. An updated list of identified substances is shown. Typical Maillard reaction products as found in warmed over flavour as aldehydes, aromatics, heterocyclic and sulphur compounds were detected. Quantification of some toxicological relevant substances is presented. The amounts of these substances are given in relation to the laser parameters and different tissues for further toxicological assessment.

  17. 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

  18. Quantification of molecular diffusion in arterial tissues with optical coherence tomography and fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Ghosn, M. G.; Syed, S. H.; Befrui, N. A.; Leba, M.; Vijayananda, A.; Sudheendran, N.; Larin, K. V.

    2009-06-01

    Alternations in vascular permeability for different molecules, drugs, and contrast agents might be a significant early marker of development of various diseases such as atherosclerosis. However, up to date experimental studies of molecular diffusion across vascular wall have been limited. Recently, we demonstrated that the Optical Coherence Tomography (OCT) technique could be applied for noninvasive and nondestructive quantification of molecular diffusion in different biological tissues. However, the viability of the OCT-based assessment of molecular diffusion should be validated with established methods. This study focused on comparing molecular diffusion rates in vascular tissues measured with OCT and standard fluorescent microscopy. Noninvasive quantification of tetramethylrhodamine (fluorescent dye) permeability in porcine vascular tissues was performed using a fiber-based OCT system. Concurrently, standard histological examination of dye diffusion was performed and quantified with fluorescent microscopy. The permeability of tetramethylrhodamine was found to be (2.08 ± 0.31) × 10-5 cm/s with the fluorescent technique ( n = 8), and (2.45 ± 0.46) × 10-5 cm/s with the OCT ( n = 3). Good correlation between permeability rates measured by OCT and histology was demonstrated, suggesting that the OCT-based method could be used for accurate, nondestructive assessment of molecular diffusion in multilayered tissues.

  19. Advantages of a dual-tracer model over reference tissue models for binding potential measurement in tumors

    NASA Astrophysics Data System (ADS)

    Tichauer, K. M.; Samkoe, K. S.; Klubben, W. S.; Hasan, T.; Pogue, B. W.

    2012-10-01

    The quantification of tumor molecular expression in vivo could have a significant impact for informing and monitoring emerging targeted therapies in oncology. Molecular imaging of targeted tracers can be used to quantify receptor expression in the form of a binding potential (BP) if the arterial input curve or a surrogate of it is also measured. However, the assumptions of the most common approaches (reference tissue models) may not be valid for use in tumors. In this study, the validity of reference tissue models is investigated for use in tumors experimentally and in simulations. Three different tumor lines were grown subcutaneously in athymic mice and the mice were injected with a mixture of an epidermal growth factor receptor-targeted fluorescent tracer and an untargeted fluorescent tracer. A one-compartment plasma input model demonstrated that the transport kinetics of both tracers was significantly different between tumors and all potential reference tissues, and using the reference tissue model resulted in a theoretical underestimation in BP of 50% ± 37%. On the other hand, the targeted and untargeted tracers demonstrated similar transport kinetics, allowing a dual-tracer approach to be employed to accurately estimate BP (with a theoretical error of 0.23% ± 9.07%). These findings highlight the potential for using a dual-tracer approach to quantify receptor expression in tumors with abnormal hemodynamics, possibly to inform the choice or progress of molecular cancer therapies.

  20. Extraction, Identification, and Quantification of Histones from Small Quantities of Specific Brain Tissue.

    PubMed

    Beldjoud, Hassiba; Messanvi, Fany; Nadif Kasri, Nael; Roozendaal, Benno

    2016-01-01

    Histone posttranslational modifications (PTMs), by their action on the chromatin state, play a central role in the regulation of gene expression. The discovery that some PTMs in the brain are dynamically regulated by experience and environmental factors makes them an important subject for the study of plasticity changes in learning and memory, addiction, and psychiatric disorders. Current histone isolation protocols, however, require large amounts of tissue, which limits their application for analyzing small tissue samples from a specific brain region. We describe here a step-by-step protocol for histone extraction and isolation from 1 mm(3) of tissue from brain punches, which allows reproducible and reliable results for histone PTM identification and quantification without losing anatomical precision. © 2016 by John Wiley & Sons, Inc. PMID:27367963

  1. Comparison of five endogenous reference genes for specific PCR detection and quantification of Brassica napus.

    PubMed

    Wu, Gang; Zhang, Li; Wu, Yuhua; Cao, Yinglong; Lu, Changming

    2010-03-10

    Five previously reported Brassica napus endogenous reference genes, including acetyl-CoA carboxylase gene (BnACCg8), phosphoenolpyruvate carboxylase (PEP), oleoyl hydrolase gene (FatA), high-mobility-group protein I/Y gene (HMG-I/Y) and cruciferin A gene (CruA), were analyzed for their PCR specificity between B. napus and other species and the quantification stability among different B. napus cultivars. PCR and sequencing results indicated that none of these systems was species-specific as required by the genetically modified organism labeling policy. When these genes were employed in real-time PCR, BnACCg8 and HMG-I/Y systems showed relatively greater heterogeneity among 10 different cultivars. The sequencing results showed that the single nucleotide polymorphism in the primer binding sites was the potential source of the instability in the HMG-I/Y system. The bias of BnACCg8 was thought to be associated with the inconsistent copy number of this gene. PMID:20143854

  2. Accurate quantification of water-macromolecule exchange induced frequency shift: effects of reference substance.

    PubMed

    Leutritz, Tobias; Hilfert, Liane; Smalla, Karl-Heinz; Speck, Oliver; Zhong, Kai

    2013-01-01

    Water-macromolecule exchange induces a bulk water frequency shift contributing to the contrast in phase imaging. For separating the effects of the water-macromolecule exchange and the macromolecule susceptibility, appropriate internal or external references are needed. In this study, two internal reference compounds, 2,2,3,3-tetradeuterio-3-trimethylsilyl-propionate (TMSP) and 1,4-dioxane, were used to study the macromolecule-dependent water frequency shift in a bovine serum albumin (BSA)-water system in detail. For TMSP, the water-macromolecule exchange shift depended on both the BSA and the reference concentration and stabilized to a value of 0.025 ppm/mM (298 K, TMSP concentrations > 30 mM). For dioxane, the dependency of the water-macromolecule exchange shift on the BSA concentration is independent of dioxane at low concentrations. The resulting shift was smaller (0.009 ppm/mM) when compared with using higher TMSP concentrations as reference. This discrepancy might be due to additional dioxane-water interactions. Measurements with an external chloroform reference in a coaxial geometry showed a shift of -0.013 ppm/mM resulting from the opposing effects of macromolecules in water exchange-induced shift and diamagnetic susceptibility shift. All these effects should be considered in the interpretation of tissue phase contrast. From the experimental data, the equilibrium binding constant between BSA and TMSP has been quantified to be K(d) = 1.3 ± 0.4, and the estimated number of interaction sites for BSA is 12.7 ± 2.6. PMID:22374834

  3. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

    PubMed Central

    Nygard, Ann-Britt; Jørgensen, Claus B; Cirera, Susanna; Fredholm, Merete

    2007-01-01

    Background Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR. Results In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (ACTB), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ, SDHA, B2M and GAPDH. Conclusion Expression stability varies greatly between genes. ACTB, RPL4, TPB and HPRT1 were found to have the highest stability across tissues. Based on both expression stability and expression level, our data suggest that ACTB and RPL4 are good reference genes for high abundant transcripts while TPB and HPRT1 are good reference genes for low abundant transcripts in expression studies across different pig tissues. PMID:17697375

  4. Quantification of ligand PET studies using a reference region with a displaceable fraction: application to occupancy studies with [11C]-DASB as an example

    PubMed Central

    Turkheimer, Federico E; Selvaraj, Sudhakar; Hinz, Rainer; Murthy, Venkatesha; Bhagwagar, Zubin; Grasby, Paul; Howes, Oliver; Rosso, Lula; Bose, Subrata K

    2012-01-01

    This paper aims to build novel methodology for the use of a reference region with specific binding for the quantification of brain studies with radioligands and positron emission tomography (PET). In particular: (1) we introduce a definition of binding potential BPD=DVR−1 where DVR is the volume of distribution relative to a reference tissue that contains ligand in specifically bound form, (2) we validate a numerical methodology, rank-shaping regularization of exponential spectral analysis (RS-ESA), for the calculation of BPD that can cope with a reference region with specific bound ligand, (3) we demonstrate the use of RS-ESA for the accurate estimation of drug occupancies with the use of correction factors to account for the specific binding in the reference. [11C]-DASB with cerebellum as a reference was chosen as an example to validate the methodology. Two data sets were used; four normal subjects scanned after infusion of citalopram or placebo and further six test–retest data sets. In the drug occupancy study, the use of RS-ESA with cerebellar input plus corrections produced estimates of occupancy very close the ones obtained with plasma input. Test–retest results demonstrated a tight linear relationship between BPD calculated either with plasma or with a reference input and high reproducibility. PMID:21811290

  5. Quantification of ligand PET studies using a reference region with a displaceable fraction: application to occupancy studies with [(11)C]-DASB as an example.

    PubMed

    Turkheimer, Federico E; Selvaraj, Sudhakar; Hinz, Rainer; Murthy, Venkatesha; Bhagwagar, Zubin; Grasby, Paul; Howes, Oliver; Rosso, Lula; Bose, Subrata K

    2012-01-01

    This paper aims to build novel methodology for the use of a reference region with specific binding for the quantification of brain studies with radioligands and positron emission tomography (PET). In particular: (1) we introduce a definition of binding potential BP(D)=DVR-1 where DVR is the volume of distribution relative to a reference tissue that contains ligand in specifically bound form, (2) we validate a numerical methodology, rank-shaping regularization of exponential spectral analysis (RS-ESA), for the calculation of BP(D) that can cope with a reference region with specific bound ligand, (3) we demonstrate the use of RS-ESA for the accurate estimation of drug occupancies with the use of correction factors to account for the specific binding in the reference. [(11)C]-DASB with cerebellum as a reference was chosen as an example to validate the methodology. Two data sets were used; four normal subjects scanned after infusion of citalopram or placebo and further six test-retest data sets. In the drug occupancy study, the use of RS-ESA with cerebellar input plus corrections produced estimates of occupancy very close the ones obtained with plasma input. Test-retest results demonstrated a tight linear relationship between BP(D) calculated either with plasma or with a reference input and high reproducibility. PMID:21811290

  6. 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

  7. Quantification of danofloxacin and difloxacin in chicken tissues in the presence of sarafloxacin as interference.

    PubMed

    Rodríguez Cáceres, M I; Guiberteau Cabanillas, A; Bohoyo Gil, D; Martínez Cañas, M A

    2009-09-01

    A new spectrofluorimetric method has been developed for the quantification of danofloxacin (DANO) and difloxacin (DIFLO), in the presence of the primary metabolite of difloxacin, with sarafloxacin (SARA) as interference, in chicken tissue samples. The method is based on second-order multivariate calibration, applying parallel factor analysis (PARAFAC), to the excitation-emission matrices (EEMs) of these compounds. High overlapping of the signals and influence of matrix effects were observed. To solve the problem, the standard addition method was used. Chemical variables were optimized. The measured EEMs of the analytes, as analytical signals, allowed their quantification in chicken tissue samples. Solid phase extraction was used for the extraction of the analytes in real samples. The range of concentration examined varied from 30 to 100 ng g(-1) for danofloxacin, and from 100 to 200 ng g(-1) for difloxacin. Both analytes can be analyzed individually, and the binary mixture can be resolved, with recoveries comprising between 88.7 and 106.6%. PMID:19689131

  8. 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.

  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. 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. PMID:26372538

  11. 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

  12. Drug detection and quantification directly from tissue using novel ionization methods for mass spectrometry.

    PubMed

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

    2015-01-01

    Solvent assisted ionization inlet (SAII) and matrix assisted ionization vacuum (MAIV) were used to quantify rapidly 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 the detection of endogenous lipids, metabolites, and clozapine directly from sections of mouse brain tissue. A rapid surface assessment was achieved by SAII with the assistance of heat applied to the mass spectrometer inlet. MAIV provided an improved reproducibility without the need of a heated inlet. In addition, isotope dilution and standard addition were used without sample clean-up, and the results correlate well with liquid chromatography tandem MS using sample work-up. Using the simple surface methods, standard solutions containing clozapine and a deuterated internal standard (clozapine-d8) at different concentration ratios were used in the extraction and quantification of clozapine from brain tissue sections of a drug-treated mouse using different tissue thicknesses. The amount of clozapine extracted by these surface methods was independent of tissue thickness. PMID:26307700

  13. 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.

  14. Consistent, multi-instrument single tube quantification of CD20 in antibody bound per cell based on CD4 reference.

    PubMed

    Degheidy, Heba; Abbasi, Fatima; Mostowski, Howard; Gaigalas, Adolfas K; Marti, Gerald; Bauer, Steven; Wang, Lili

    2016-03-01

    Detecting changes in the expression levels of cell antigens could provide critical information for the diagnosis of many diseases, for example, leukemia, lymphoma, and immunodeficiency diseases, detecting minimal residual disease, monitoring immunotherapies and discovery of meaningful clinical disease markers. One of the most significant challenges in flow cytometry is how to best ensure measurement quality and generate consistent and reproducible inter-laboratory and intra-laboratory results across multiple cytometer platforms and locations longitudinally over time. In a previous study, we developed a procedure for instrument standardization across four different flow cytometer platforms from the same manufacturer. CD19 quantification was performed on three of the standardized instruments relative to CD4 expression on T lymphocytes with a known amount of antibody bound per cell (ABC) as a quantification standard. Consistent and reliable measures of CD19 expression were obtained independent of fluorochrome used demonstrating the utility of this approach. In the present investigation, quantification of CD20 relative to CD4 reference marker was implemented within a single tube containing both antibodies. Relative quantification of CD20 was performed using anti-CD20 antibody (clone L27) in three different fluorochromes relative to anti-CD4 antibody (clone SK3). Our results demonstrated that cell surface marker quantification can be performed robustly using the single tube assay format with novel gating strategies. The ABC values obtained for CD20 expression levels using PE, APC, or PerCP Cy5.5 are consistent over the five different instrument platforms for any given apparently healthy donor independent of the fluorochrome used. PMID:26013593

  15. 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

  16. Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

    PubMed Central

    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

  17. 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.

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

    PubMed Central

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

    2012-01-01

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

  19. 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

  20. 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

  1. 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

  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. 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

  4. 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.

  5. Brown Adipose Tissue Quantification in Human Neonates Using Water-Fat Separated MRI

    PubMed Central

    Rasmussen, Jerod M.; Entringer, Sonja; Nguyen, Annie; van Erp, Theo G. M.; Guijarro, Ana; Oveisi, Fariba; Swanson, James M.; Piomelli, Daniele; Wadhwa, Pathik D.

    2013-01-01

    There is a major resurgence of interest in brown adipose tissue (BAT) biology, particularly regarding its determinants and consequences in newborns and infants. Reliable methods for non-invasive BAT measurement in human infants have yet to be demonstrated. The current study first validates methods for quantitative BAT imaging of rodents post mortem followed by BAT excision and re-imaging of excised tissues. Identical methods are then employed in a cohort of in vivo infants to establish the reliability of these measures and provide normative statistics for BAT depot volume and fat fraction. Using multi-echo water-fat MRI, fat- and water-based images of rodents and neonates were acquired and ratios of fat to the combined signal from fat and water (fat signal fraction) were calculated. Neonatal scans (n = 22) were acquired during natural sleep to quantify BAT and WAT deposits for depot volume and fat fraction. Acquisition repeatability was assessed based on multiple scans from the same neonate. Intra- and inter-rater measures of reliability in regional BAT depot volume and fat fraction quantification were determined based on multiple segmentations by two raters. Rodent BAT was characterized as having significantly higher water content than WAT in both in situ as well as ex vivo imaging assessments. Human neonate deposits indicative of bilateral BAT in spinal, supraclavicular and axillary regions were observed. Pairwise, WAT fat fraction was significantly greater than BAT fat fraction throughout the sample (ΔWAT-BAT = 38%, p<10−4). Repeated scans demonstrated a high voxelwise correlation for fat fraction (Rall = 0.99). BAT depot volume and fat fraction measurements showed high intra-rater (ICCBAT,VOL = 0.93, ICCBAT,FF = 0.93) and inter-rater reliability (ICCBAT,VOL = 0.86, ICCBAT,FF = 0.93). This study demonstrates the reliability of using multi-echo water-fat MRI in human neonates for quantification throughout the torso of BAT depot

  6. 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

  7. 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

  8. 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. PMID:26553206

  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. 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).

  11. Identification of Reference Genes for Relative Quantification of Circulating MicroRNAs in Bovine Serum

    PubMed Central

    Bae, In-Seon; Chung, Ki Yong; Yi, Jongmin; Kim, Tae Il; Choi, Hwa-Sik; Cho, Young-Moo; Choi, Inho; Kim, Sang Hoon

    2015-01-01

    Circulating microRNAs in body fluids have been implicated as promising biomarkers for physiopathology disorders. Currently, the expression levels of circulating microRNAs are estimated by reverse transcription quantitative real-time polymerase chain reaction. Use of appropriate reference microRNAs for normalization is critical for accurate microRNA expression analysis. However, no study has systematically investigated reference genes for evaluating circulating microRNA expression in cattle. In this study, we describe the identification and characterization of appropriate reference microRNAs for use in the normalization of circulating microRNA levels in bovine serum. We evaluated the expression stability of ten candidate reference genes in bovine serum by using reverse transcription quantitative real-time polymerase chain reaction. Data were analyzed using geNorm, NormFinder, and BestKeeper statistical algorithms. The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference. The suitability of these microRNAs was validated, and even when compared among different genders or breeds, the combination of miR-93 and miR-127 was ranked as the most stable microRNA reference. Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum. The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle. PMID:25826387

  12. 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

  13. Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis

    PubMed Central

    Eissa, Nour; Hussein, Hayam; Wang, Hongxing; Rabbi, Mohammad F.; Bernstein, Charles N.

    2016-01-01

    Background Many animal models have been developed to characterize the complexity of colonic inflammation. In dextran sodium sulfate (DSS) experimental colitis in mice the choice of reference genes is critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR). No studies have addressed the performance of reference genes in mice DSS-experimental colitis. This study aimed to determine the stability of reference genes expression (RGE) in DSS-experimental murine colitis. Methods Colitis was induced in male C57BL/6 mice using DSS5% for 5 days, control group received water. RNA was extracted from inflamed and non-inflamed colon. Using RT-qPCR, comparative analysis of 13 RGE was performed according to predefined criteria and relative colonic TNF-α and IL-1β gene expression was determined by calculating the difference in the threshold cycle. Results Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest variability within the inflamed and control groups. Conversely, TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. Normalization of colonic TNF-α and IL-1β mRNA levels was dependent on the reference gene used. Depending on the genes used to normalize the data, statistical significance varied from significant when TBP / Eef2 were used to non-significant when Gapdh, Actb or β2m were used. Conclusions This study highlights the appropriate choice of RGE to ensure adequate normalization of RT-qPCR data when using this model. Suboptimal RGE may explain controversial results from published studies. We recommend using Tbp and Eef2 instead of Gapdh, Actb or β2m as reference genes. PMID:27244258

  14. 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

  15. 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

  16. 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

  17. Performance of automated platelet quantification using different analysers in comparison with an immunological reference method in thrombocytopenic patients

    PubMed Central

    Trabuio, Ernesto; Valverde, Sara; Antico, Francesco; Manoni, Fabio; Gessoni, Gianluca

    2009-01-01

    Background Rapidly available and accurate platelet counts play an important role in the evaluation of haemorrhagic status and in assessing the need for platelet transfusions. We, therefore, evaluated platelet counting performance of haematology analysers using optical, impedance and immunological methods in thrombocytopenic patients. Materials and Methods We considered 99 patients with a platelet (plt) count under 50x109 plt/L. We compared the platelet counts obtained using ADVIA 2120 (optical method), Cell-Dyn Sapphire (optical, impedance and immunological methods with CD61) and a reference, double staining (CD41+CD61) immunological method. Results The platelet counts of all the considered methods showed good correlation with those of the reference method, despite an overestimation in platelet quantification. The degree of inaccuracy was greater for platelet counts under 20 x109 plt/L. Conclusions Clinicians who use platelet thresholds below 20 x109 plt/L for making clinical decisions must be aware of the limitations in precision and accuracy of cell counters at this level of platelet count. Inaccurate counts of low platelet numbers could create problems if attempts are made to reduce the threshold below 20x109 plt/L. PMID:19290080

  18. Defect quantification with reference-free thermal contrast and artificial neural networks

    NASA Astrophysics Data System (ADS)

    Benitez, Hernan D.; Ibarra-Castanedo, Clemente; Bendada, AbdelHakim; Maldague, Xavier; Loaiza, Humberto; Caicedo, Eduardo

    2007-04-01

    The Infrared Nondestructive Testing (IRNT) methods based on thermal contrast are strongly affected by non-uniform heating at the surface. Hence, the results obtained from these methods considerably depend on the chosen reference point. One of these methods is Artificial Neural Networks (ANN) that uses thermal contrast curves as input data for training and test in order to detect and estimate defect depth. The Differential Absolute Contrast (DAC) has been successfully used as an alternative thermal contrast to eliminate the need of a reference point by defining the thermal contrast with respect to an ideal sound area. The DAC technique has been proven effective to inspect materials at early times since it is based on the 1D solution of the Fourier equation. A modified DAC version using thermal quadrupoles explicitly includes the sample thickness in the solution, extending in this way the range of validity when the heat front approaches the sample rear face. We propose to use ANN to detect and quantify defects in composite materials using data extracted from the modified DAC with thermal quadrupoles in order to decrease the non-uniform heating and plate shape impact on the inspection.

  19. 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. PMID:23411239

  20. Correlation of virtual touch tissue quantification and liver biopsy in a rat liver fibrosis model.

    PubMed

    Hu, Zhiwen; Luo, Jialun; Wei, Hongqin; Ou, Wencai; Xiao, Shuyi; Gan, Man; Ma, Suihong; He, Jingguang; Wu, Daihong; Feng, Guiying; Wei, Jinglu; Liu, Jianhua

    2015-05-01

    Liver fibrosis assessment is very important to the treatment of chronic liver disease. In the present study, Virtual Touch Tissue Quantification (VTQ) and eSie Touch™ elasticity imaging techniques were used to examine the rat liver fibrosis model. Rat liver fibrosis was induced with thioacetamide and the degree of liver fibrosis was determined using pathological diagnosis as a gold standard. The right lobe of the liver was also examined with the VTQ and eSie Touch™ techniques. The VTQ and serological results were correlated and analyzed. The results were compared with those obtained from liver biopsies to investigate the accuracy and diagnostic value of eSie Touch™ and VTQ on the classification of liver fibrosis in rats. A total of 30 successful modeling cases were obtained, with a success rate of 86%. The mean acoustic radiation force impulse (ARFI) elastography‑VTQ values were 1.08, 1.51, 1.88 and 2.50 m/sec for the normal and F1/F2, F3 and F4 fibrosis groups, respectively. A significant correlation (r = 0.969) was identified between the ARFI measurements and the degree of fibrosis assessed by pathological examination (P<0.001). The histological staging results correlated with those of the eSie Touch™ elasticity imaging of the biopsy site (r = 0.913, P<0.001). The predictive values of ARFI for various stages of fibrosis were as follows: F≥1 and 2 ‑ cut‑off >1.250 m/sec (when Vs >1.250 m/sec, the pathological grading was ≥F1/F2) [Area under receiver operating characteristic (AUROC) = 1.00], F≥3 ‑ cut‑off >1.685 m/sec (when Vs >1.685 m/sec, the pathological grading was ≥F3; AUROC = 1.00) and F≥4 ‑ cut‑off >2.166 m/sec (when Vs >2.166 m/sec, the pathological grading is cirrhosis; AUROC = 1.00). In conclusion, the eSie Touch™ elasticity imaging and VTQ techniques may be successfully adopted to assess the extent of liver stiffness. These techniques are expected to replace liver biopsy. PMID:25592825

  1. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25691256

  3. 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

  4. 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

  5. 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.

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

    PubMed

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

    2016-01-01

    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

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

    PubMed Central

    2014-01-01

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

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

    PubMed

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

    2014-10-21

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

  9. 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

  10. 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

  11. 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. PMID:25689595

  12. 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

  13. 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

  14. A spiked tissue-based approach for quantification of phosphatidylcholines in brain section by MALDI mass spectrometry imaging.

    PubMed

    Jadoul, Laure; Longuespée, Rémi; Noël, Agnès; De Pauw, Edwin

    2015-03-01

    In the last few years, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has been successfully used to study the distribution of lipids within tissue sections. However, few efforts have been made to acquire reliable quantitative data regarding the localized concentrations of these molecules. Here we propose an approach based on brain homogenates for the quantification of phosphatidylcholines (PCs) in brain section by MALDI MSI. Homogenates were spiked with a range of PC(16:0 d31/18:1) concentrations. Sections from homogenates and intact brain were simultaneously prepared before being analyzed by MALDI MSI using a Fourier transform ion cyclotron resonance (FT-ICR) analyzer. Standard curves were generated from the signal intensity of the different PC(16:0 d31/18:1) ionic species ([M+H](+), [M+Na](+) and [M+K](+)) detected from the homogenate sections. Localized quantitative data were finally extracted by correlating the standard curves with the signal intensities of endogenous PC (especially PC(16:0/18:1)) ionic species detected on different areas of the brain section. They were consistent with quantitative values found in the literature. This work introduces a new method to take directly into account biological matrix effects for the quantification of lipids as well as other endogenous compounds, in tissue sections by MALDI MSI. PMID:25326885

  15. 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

  16. 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

  17. 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

  18. A Quantification Method for Breast Tissue Thickness and Iodine Concentration Using Photon-Counting Detector.

    PubMed

    Han, Seokmin

    2015-10-01

    The purpose of contrast-enhanced digital mammography (CEDM) is to facilitate detection and characterization of the lesions in the breast using intravenous injection of an iodinated contrast agent. CEDM produces iodine images with gray levels proportional to iodine concentration at each pixel, which can be considered as quantification of iodine. While dual-energy CEDM requires an accurate knowledge of the thickness of compressed breast for the quantification, it is known that the accuracy of the built-in thickness measurement is not satisfactory. Triple-energy CEDM, which can provide a third image, can alleviate the limitation of dual-energy CEDM. If triple exposure technique is applied, it can lead to increased risk of motion artifact. An energy-resolving photon-counting detector (PCD) that can acquire multispectral X-ray images can reduce the risk of motion artifact. In this research, an easily implementable method for iodine quantification in breast imaging was suggested, and it was applied to the images of breast phantom with various iodine concentrations. The iodine concentrations in breast phantom simulate lesions filled with different iodine concentrations in the breast. The result shows that the proposed method can quantify the iodine concentrations in breast phantom accurately. PMID:25708894

  19. Quantification of the impact of MLC modeling and tissue heterogeneities on dynamic IMRT dose calculations

    SciTech Connect

    Mihaylov, I. B.; Lerma, F. A.; Fatyga, M.; Siebers, J. V.

    2007-04-15

    This study quantifies the dose prediction errors (DPEs) in dynamic IMRT dose calculations resulting from (a) use of an intensity matrix to estimate the multi-leaf collimator (MLC) modulated photon fluence (DPE{sub IGfluence}) instead of an explicit MLC particle transport, and (b) handling of tissue heterogeneities (DPE{sub hetero}) by superposition/convolution (SC) and pencil beam (PB) dose calculation algorithms. Monte Carlo (MC) computed doses are used as reference standards. Eighteen head-and-neck dynamic MLC IMRT treatment plans are investigated. DPEs are evaluated via comparing the dose received by 98% of the GTV (GTV D{sub 98%}), the CTV D{sub 95%}, the nodal D{sub 90%}, the cord and the brainstem D{sub 02%}, the parotid D{sub 50%}, the parotid mean dose (D{sub Mean}), and generalized equivalent uniform doses (gEUDs) for the above structures. For the MC-generated intensity grids, DPE{sub IGfluence} is within {+-}2.1% for all targets and critical structures. The SC algorithm DPE{sub hetero} is within {+-}3% for 98.3% of the indices tallied, and within {+-}3.4% for all of the tallied indices. The PB algorithm DPE{sub hetero} is within {+-}3% for 92% of the tallied indices. Statistical equivalence tests indicate that PB DPE{sub hetero} requires a {+-}3.6% interval to state equivalence with the MC standard, while the intervals are <1.5% for SC DPE{sub hetero} and DPE{sub IGfluence}. Overall, these results indicate that SC and MC IMRT dose calculations which use MC-derived intensity matrices for fluence prediction do not introduce significant dose errors compared with full Monte Carlo dose computations; however, PB algorithms may result in clinically significant dose deviations.

  20. Fluorescence spectroscopy using excitation and emission matrix for quantification of tissue native fluorophores and cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Gayen, S. K.; Xu, M.

    2014-03-01

    Native fluorescence spectrum of normal and cancerous human prostate tissues is studied to distinguish between normal and cancerous tissues, and cancerous tissues at different cancer grade. The tissue samples were obtained from Cooperative Human Tissue Network (CHTN) and National Disease Research Interchange(NDRI). An excitation and emission matrix (EEM) was generated for each tissue sample by acquiring native fluorescence spectrum of the sample using multiple excitation wavelengths. The non-negative matrix factorization algorithm was used to generate fluorescence EEMs that correspond to the fluorophores in biological tissues, including tryptophan, collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and the background paraffin. We hypothesize that, as a consequence of metabolic changes associated with the development of cancer, the concentrations of NADH and FAD are different in normal and cancerous tissues, and also different for different cancer grades. We used the ratio of the abundances of FAD and NADH to distinguish between normal and cancerous tissues, and the tissue cancer grade. The FAD-to-NADH ratio was found to be the highest for normal tissue and decreased as the cancer grade increased.

  1. 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

  2. Construction of Reference Data for Tissue Characterization of Arterial Wall Based on Elasticity Images

    NASA Astrophysics Data System (ADS)

    Inagaki, Jun; Hasegawa, Hideyuki; Kanai, Hiroshi; Ichiki, Masataka; Tezuka, Fumiaki

    2005-06-01

    Previously, we developed the phased tracking method [H. Kanai et al.: IEEE Trans. Ultrason. Ferroelectr. Freq. Control 43 (1996) 791] for measuring the minute change in thickness during one heartbeat and the elasticity of the arterial wall. By comparing pathological images with elasticity images measured with ultrasound, elasticity distributions for respective tissues in the arterial wall were determined. We have already measured the elasticity distributions for lipids and fibrous tissues (mixtures of smooth-muscle and collagen fiber) [H. Kanai et al.: Circulation 107 (2003) 3018]. In this study, elasticity distributions were measured for blood clots and calcified tissues. We discuss whether these elasticity distributions, which were measuerd in vitro, can be used as reference data for classifying cross-sectional elasticity images measured in vivo into respective tissues. In addition to the measurement of elasticity distributions, correlations between collagen content and elasticity were investigated with respect to fibrous tissue to estimate the collagen and smooth-muscle content based on elasticity. Collagen and smooth-muscle content may be important factors in determining the stability of the fibrous cap of atherosclerotic plaque. Therefore, correlations between elasticity and elements of the tissue in the arterial wall may provide useful information for the noninvasive diagnosis of plaque vulnerability.

  3. Development of a Novel Reference Plasmid for Accurate Quantification of Genetically Modified Kefeng6 Rice DNA in Food and Feed Samples

    PubMed Central

    Zhang, Xiujie; Wan, Yusong

    2013-01-01

    Reference plasmids are an essential tool for the quantification of genetically modified (GM) events. Quantitative real-time PCR (qPCR) is the most commonly used method to characterize and quantify reference plasmids. However, the precision of this method is often limited by calibration curves, and qPCR data can be affected by matrix differences between the standards and samples. Here, we describe a digital PCR (dPCR) approach that can be used to accurately measure the novel reference plasmid pKefeng6 and quantify the unauthorized variety of GM rice Kefeng6, eliminating the issues associated with matrix effects in calibration curves. The pKefeng6 plasmid was used as a calibrant for the quantification of Kefeng6 rice by determining the copy numbers of event- (77 bp) and taxon-specific (68 bp) fragments, their ratios, and their concentrations. The plasmid was diluted to five different concentrations. The third sample (S3) was optimized for the quantification range of dPCR according to previous reports. The ratio between the two fragments was 1.005, which closely approximated the value certified by sequencing, and the concentration was found to be 792 copies/μL. This method was precise, with an RSD of ~3%. These findings demonstrate the advantages of using the dPCR method to characterize reference materials. PMID:24324952

  4. 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

  5. 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

    2015-01-01

    The objective of this study was to evaluate the accuracy of dual-energy CT (DECT) for quantifying iodine using a soft tissue-mimicking phantom across various DECT acquisition parameters and dual-source CT (DSCT) scanners. A phantom was constructed with plastic tubes containing soft tissue-mimicking materials with known iodine concentrations (0-20 mg/mL). Experiments were performed on two DSCT scanners, one equipped with an integrated detector and the other with a conventional detector. DECT data were acquired using two DE modes (80 kV/Sn140 kV and 100 kV/Sn140 kV) with four pitch values (0.6, 0.8, 1.0, and 1.2). Images were reconstructed using a soft tissue kernel with and without beam hardening correction (BHC) for iodine. Using the dedicated DE software, iodine concentrations were measured and compared to true concentrations. We also investigated the effect of reducing gantry rotation time on the DECT-based iodine measurement. At iodine concentrations higher than 10 mg/mL, the relative error in measured iodine concentration increased slightly. This error can be decreased by using the kernel with BHC, compared with the kernel without BHC. Both 80 kV/Sn140 kV and 100 kV/Sn140 kV modes could provide accurate quantification of iodine content. Increasing pitch value or reducing gantry rotation time had only a minor impact on the DECT-based iodine measurement. The DSCT scanner, equipped with the new integrated detector, showed more accurate iodine quantification for all iodine concentrations higher than 10 mg/mL. An accurate quantification of iodine can be obtained using the second-generation DSCT scanner in various DE modes with pitch values up to 1.2 and gantry rotation time down to 0.28 s. For iodine concentrations ≥ 10 mg/mL, using the new integrated detector and the kernel with BHC can improve the accuracy of DECT-based iodine measurements. PMID:26699312

  6. Method for non-optical quantification of in situ local soft tissue biomechanics.

    PubMed

    Tarsi, Grant M; Gould, Russell A; Chung, Jaebum A; Xu, Andrew Z; Bozkurt, Alper; Butcher, Jonathan T

    2013-07-26

    Soft tissues exhibit significant biomechanical changes as they grow, adapt, and remodel under a variety of normal and pathogenic stimuli. Biomechanical measurement of intact soft tissues is challenging because of its large strain and nonlinear behavior. Tissue distention through applied vacuum pressure is an attractive method for acquiring local biomechanical information minimally invasive and non-destructive, but the current requirement for optical strain measurement limits its use. In this study, we implemented a novel flexible micro-electrode array placed within a cylindrical probe tip. We hypothesized that upon tissue distention, contact with each electrode would result in a precipitous voltage drop (from the resistive connection formed between input and output electrodes) across the array. Hence, tissue distention (strain) can be derived directly from the electrode array geometry. In pilot studies, we compared the electrode array measurements directly against optical deformation measurements in-situ of agar tissue phantoms and freshly isolated porcine tissue. Our results demonstrate that the probe derived stress-strain profiles and modulus measurements were statistically indistinguishable from optical measurement. We further show that electrode geometry can be scaled down to 50μm in size (length and width) and spaced 50μm apart without impairing measurement accuracy. These results establish a promising new method for minimally invasive local soft tissue biomechanical measurement, which may be useful for applications such as disease diagnosis and health monitoring. PMID:23791186

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

    PubMed Central

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

    2011-01-01

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

  8. 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. PMID:25078400

  9. 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.

  10. PCR-Based quantification of Borrelia burgdorferi organisms in canine tissues over a 500-Day postinfection period.

    PubMed

    Straubinger, R K

    2000-06-01

    Borrelia burgdorferi infection in beagle dogs was studied quantitatively with skin punch biopsy samples and blood samples collected at 4- and 2-week intervals, respectively, over a 500-day period. Thereafter, 25 tissue samples of each dog were collected for further analysis. Starting at day 120 after tick challenge, 12 dogs were treated with antibiotics (azithromycin, ceftriaxone, or doxycycline) for 30 consecutive days. Four dogs received no antibiotic therapy. Quantification of B. burgdorferi DNA was done with an ABI Prism 7700 Sequence Detection System with oligonucleotide primers and a fluorescence-labeled probe designed to specifically amplify a fragment of the ospA gene of B. burgdorferi strain N40. All 16 dogs became infected with B. burgdorferi after tick challenge. In skin biopsy samples, spirochete numbers peaked at day 60 postinfection (<1.5 x 10(6) organisms per 100 microgram of extracted DNA), at the same time when clinical signs of arthritis developed in 11 of 16 dogs, and decreased to almost undetectable levels during the following 6 months. The number of B. burgdorferi organisms detected in skin biopsy samples was inversely correlated with the antibody levels measured by enzyme-linked immunosorbent assay. Antibiotic treatment reduced the amount of detectable spirochete DNA in skin tissue by a factor of 1,000 or more. At the end of the experiment, B. burgdorferi DNA was detectable at low levels (10(2) to 10(4) organisms per 100 microgram of extracted DNA) in multiple tissue samples regardless of treatment. However, more tissue samples of untreated dogs than of antibiotic-treated dogs were positive, and tissue samples of untreated dogs also were positive by culture. Only 1.6% of 576 blood samples of all dogs were positive for B. burgdorferi by PCR. PMID:10834975

  11. 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. PMID:26939754

  12. 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. PMID:26678179

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

    PubMed

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

    2015-02-01

    The digital separation of diaminobenzidine (DAB)-stained tissues from hematoxylin background is an important pre-processing step to analyze immunostains. In most stain separation methods, specific color channels (for example: RGB, HSI, CMYK) or color deconvolution matrices are used to obtain different tissue contrasts between DAB- and hematoxylin-stained areas. However, these methods could produce incomplete separation or color changes because the color spectra of stains and co-localized stains overlap in histological images. Therefore, we proposed an automatic color-filtering to separate hematoxylin- and DAB-stained tissues. In implantation, the RGB images of DAB-labeled immunostains are first converted to 8-bit BN images by a mathematical translation to produce the largest contrast between brown DAB-stained tissues and blue hematoxylin-stained tissues. The first valley in the histogram revised by nonuniform quantization is set as the cut-off point to obtain a brown filter. DAB-stained tissues are accurately delineated from the background counterstain, resulting in DAB-only-image and De-DAB-image. Subsequently, a blue filter is designed in the CIE-Lab color space to further delineate the hematoxylin-stained tissues from the De-DAB-image. Finally, the average values of the remaining pixels of the De-DAB-image are set as the background color of the DAB-only-image to manage uneven dyeing and provide DAB-stained-image for adaptive immunohistochemistry quantitation. Extensive experimental results demonstrated that the proposed method has significant advantages compared with existing methods in terms of complete stain separation without changing the color in DAB-stained areas. PMID:25780744

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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...

  3. Quantification of hyaluronan and chondroitin/dermatan sulfates in the tissue sections on glass slides.

    PubMed

    Koshiishi, I; Horikoshi, E; Imanari, T

    1999-02-01

    The method for the determination of hyaluronan and chondroitin/dermatan sulfates in the tissue sections on a glass slide, which were prepared by histological technique, was established by applying to porcine skin. The degradation of these glycosaminoglycans to the unsaturated disaccharides in porcine skin sections on a glass slide was achieved by chondroitinase ABC and ACII in the presence of highly purified bacterial collagenase. Subsequently, the resulting unsaturated disaccharides were determined by HPLC with fluorometric postcolumn derivatization using 2-cyanoacetamide as a reagent. So far, the determination of the glycosaminoglycans in the tissues has taken up more than 5 days, whereas the determination of the glycosaminoglycans in the frozen sections by the present method was completed within a day. In addition, applications of the present method to the serial polyester wax sections processed with a small surgical knife made it possible to determine the glycosaminoglycans in a local part in the tissue section. The present method should open a way for the clinical analysis of glycosaminoglycans in the pathological tissue samples. PMID:9918675

  4. Quantification of ultrasonic texture intra-heterogeneity via volumetric stochastic modeling for tissue characterization

    PubMed Central

    Al-Kadi, Omar S.; Chung, Daniel Y.F.; Carlisle, Robert C.; Coussios, Constantin C.; Noble, J. Alison

    2015-01-01

    Intensity variations in image texture can provide powerful quantitative information about physical properties of biological tissue. However, tissue patterns can vary according to the utilized imaging system and are intrinsically correlated to the scale of analysis. In the case of ultrasound, the Nakagami distribution is a general model of the ultrasonic backscattering envelope under various scattering conditions and densities where it can be employed for characterizing image texture, but the subtle intra-heterogeneities within a given mass are difficult to capture via this model as it works at a single spatial scale. This paper proposes a locally adaptive 3D multi-resolution Nakagami-based fractal feature descriptor that extends Nakagami-based texture analysis to accommodate subtle speckle spatial frequency tissue intensity variability in volumetric scans. Local textural fractal descriptors – which are invariant to affine intensity changes – are extracted from volumetric patches at different spatial resolutions from voxel lattice-based generated shape and scale Nakagami parameters. Using ultrasound radio-frequency datasets we found that after applying an adaptive fractal decomposition label transfer approach on top of the generated Nakagami voxels, tissue characterization results were superior to the state of art. Experimental results on real 3D ultrasonic pre-clinical and clinical datasets suggest that describing tumor intra-heterogeneity via this descriptor may facilitate improved prediction of therapy response and disease characterization. PMID:25595523

  5. 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.

  6. 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

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

    PubMed

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

    2016-08-01

    The cuttlefish belongs to the mollusk class Cephalopoda, considered as the most advanced marine invertebrates and thus widely used as models to study the biology of complex behaviors and cognition, as well as their related neurochemical mechanisms. Surprisingly, methods to quantify the biogenic monoamines and their metabolites in cuttlefish brain remain sparse and measure a limited number of analytes. This work aims to validate an HPLC-ECD method for the simultaneous quantification of dopamine, serotonin, norepinephrine and their main metabolites in cuttlefish brain. In comparison and in order to develop a method suitable to answer both ecological and biomedical questions, the validation was also carried out on a phylogenetically remote species: mouse (mammals). The method was shown to be accurate, precise, selective, repeatable and sensitive over a wide range of concentrations for 5-hydroxyindole-3-acetic acid, serotonin, dopamine, 3,4-dihydroxyphenylacetic acid and norepinephrine in the both extracts of cuttlefish and mouse brain, though with low precision and recovery for 4-hydroxy-3-methoxyphenylethylene glycol. Homovanillic acid, accurately studied in rodents, was not detectable in the brain of cuttlefish. Overall, we described here the first fully validated HPLC method for the routine measurement of both monoamines and metabolites in cuttlefish brain. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26613377

  8. 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

  9. Iterative multiple reference tissue method for estimating pharmacokinetic parameters on prostate DCE MRI

    NASA Astrophysics Data System (ADS)

    Ginsburg, Shoshana B.; Bloch, B. Nicolas; Rofsky, Neil M.; Genega, Elizabeth M.; Lenkinski, Robert E.; Madabhushi, Anant

    2013-02-01

    Pharmacokinetic (PK) parameters are probes of tissue status that can be assessed by analysis of dynamic contrast-enhanced (DCE) MRI and are useful for prostate cancer (CaP) detection and grading. Traditionally, PK analysis requires knowledge of the time-resolved concentration of the contrast agent in the blood plasma, the arterial input function (AIF), which is typically estimated in an artery in the field-of-view (FOV). In cases when no suitable artery is present in the FOV, the multiple reference tissue method (MRTM) enables the estimation of PK parameters without the AIF by leveraging PK parameter values from the literature for a reference tissue in the FOV. Nevertheless, PK parameters estimated in the prostate vary significantly between patients. Consequently, population-based values obtained from the literature may introduce error into PK parameter estimation via MRTM. The objectives of this paper are two-fold. First we present a novel scheme, iterative MRTM (IMRTM), to estimate PK parameter values in the absence of the AIF without making assumptions about the PK constants associated with a reference tissue. Then, using IMRTM we investigate differences in PK constants between CaP in the peripheral zone (PZ) and CaP in the central gland (CG), as CG and PZ CaP have previously been shown to differ significantly in terms of both texture and prognosis. We apply IMRTM to 15 patients with CaP in either the CG or the PZ who were scheduled for a radical prostatectomy and a pre-operative MRI. Values for the PK parameters Ktrans and ve estimated via IMRTM average 0.29 and 0.60 for normal central gland (CG), 0.29 and 0.64 for normal peripheral zone (PZ), and 0.30 and 0.53 for CaP. It is noteworthy that PK constants estimated in PZ CaP are significantly higher than those estimated in CG CaP (p < 0:05). While both MRTM and IMRTM provide PK parameter values that are biologically feasible, IMRTM has the advantage that it invokes patient-specific information rather than

  10. 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.

  11. 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.

  12. 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

  13. Quantification of multiple compounds containing heterogeneous elements in the mixture by one-dimensional nuclear magnetic resonance spectroscopy of different nuclei using a single universal concentration reference.

    PubMed

    Xu, Li; Shi, Xiaohuo; Hu, Kaifeng

    2014-12-01

    One-dimensional (1D) quantitative NMR (qNMR) is a useful tool for concentration determination due to its experimental simplicity and the direct proportionality of the integrated signal area to the number of nuclei spin. For complex mixtures, however, signal overlapping often in one-dimensional quantitative (1) H NMR (1D (1) H qNMR) spectrum limits the accurate quantification of individual compound. Here, we introduced employing joint 1D qNMR methods of different nuclei, such as (1) H and (31) P (or/and (19) F), to quantify multiple compounds in a complex mixture using a single universal concentration reference. When the concentration ratio of several compounds containing different elements in a complex mixture is of interest, the result calculated from measured intensities from 1D qNMR of different nuclei is independent of the gravimetric error from the reference. In this case, the common reference also serves as a 'quantitative bridge' among these 1D qNMR of different nuclei. Quantitative analysis of choline, phosphocholine, and glycerophosphocholine mixture is given as an example using trimethylphosphine oxide ((CH(3))(3) P(O)) as concentration reference. Compounds containing multiple elements, such as tetramethylammonium hexafluorophosphate (N(+) (CH(3))(4 PF6 (-) are proposed as the common concentration reference for (1) H, (13) C, (15) N, (31) P, and (19) F qNMR for the quantitative analysis of complex mixture containing these different elements. We anticipate that the proposed joint 1D qNMR approach using a universal concentration reference will be a valuable alternative for simultaneous quantification of multiple compounds in a complex mixture due to its accuracy and single and simple sample preparation. PMID:25298349

  14. 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

  15. Quantification of fluid shear stress in bone tissue engineering scaffolds with spherical and cubical pore architectures.

    PubMed

    Zhao, Feihu; Vaughan, Ted J; McNamara, Laoise M

    2016-06-01

    Recent studies have shown that mechanical stimulation, in the form of fluid perfusion and mechanical compression, can enhance osteogenic differentiation of mesenchymal stem cells and bone cells within tissue engineering scaffolds in vitro. The precise nature of mechanical stimulation within tissue engineering scaffolds is not only dictated by the exogenously applied loading regime, but also depends on the geometric features of the scaffold, in particular architecture, pore size and porosity. However, the precise contribution of each geometric feature towards the resulting mechanical stimulation within a scaffold is difficult to characterise due to the wide range of interacting parameters. In this study, we have applied a fluid-structure interaction model to investigate the role of scaffold geometry (architecture, pore size and porosity) on pore wall shear stress (WSS) under a range of different loading scenarios: fluid perfusion, mechanical compression and a combination of perfusion and compression. It is found that scaffold geometry (spherical and cubical pores), in particular the pore size, has a significant influence on the stimulation within scaffolds. Furthermore, we observed an amplified WSS within scaffolds under a combination of fluid perfusion and mechanical compression, which exceeded that caused by individual fluid perfusion or mechanical compression approximately threefold. By conducting this comprehensive parametric variation study, an expression was generated to allow the design and optimisation of 3D TE scaffolds and inform experimental loading regimes so that a desired level of mechanical stimulation, in terms of WSS is generated within the scaffold. PMID:26224148

  16. 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. PMID:24962015

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

    PubMed

    Zhang, Dunhua; Moreira, Gabriel S A; Shoemaker, Craig; Newton, Joseph C; Xu, De-Hai

    2016-05-01

    The aim of this study was to understand the pathogenesis of motile aeromonas septicemia caused by an emergent, high virulent Aeromonas hydrophila (vAh) in channel catfish, Ictalurus punctatus Adipose fin clipped catfish were challenged with vAh using a waterborne challenge method, and the distribution of vAh over a time course was detected and quantified using real-time polymerase chain reaction. The results showed that 77.8% of fish died within 48 h post challenge with mean day to death of 1.5 days. At 2 h post challenge, vAh (inferred from genomic DNA copies or genome equivalents) was detected in all external and internal tissues sampled. Gill had the highest vAh cells at 1 h post challenge. Spleen harbored the most vAh cells among internal organs at 4 h post challenge. The tissues/organs with most vAh cells detected at 8 h post challenge were adipose fin, blood, intestine, kidney and skin, while liver showed the highest vAh cells at 24 h post challenge. These results suggest that vAh was able to rapidly proliferate and spread, following wound infection, through the fish blood circulation system and cause mortality within 8-24 h. PMID:27044300

  18. 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

  19. 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. PMID:16636447

  20. 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.

  1. 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. PMID:25335820

  2. 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

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

    PubMed Central

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

    2015-01-01

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

  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. PMID:23824526

  5. 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

  6. Quantification of neurotransmitters in mouse brain tissue by using liquid chromatography coupled electrospray tandem mass spectrometry.

    PubMed

    Kim, Tae-Hyun; Choi, Juhee; Kim, Hyung-Gun; Kim, Hak Rim

    2014-01-01

    A simple and rapid liquid chromatography tandem mass spectrometry method has been developed for the determination of BH4, DA, 5-HT, NE, EP, Glu, and GABA in mouse brain using epsilon-acetamidocaproic acid and isotopically labeled neurotransmitters as internal standards. Proteins in the samples were precipitated by adding acetonitrile, and then the supernatants were separated by a Sepax Polar-Imidazole (2.1 mm × 100 mm, i.d., 3 μm) column by adding a mixture of 10 mM ammonium formate in acetonitrile/water (75 : 25, v/v, 300 μl/min) for BH4 and DA. To assay 5-HT, NE, EP, Glu, and GABA; a Luna 3 μ C18 (3.0 mm × 150 mm, i.d., 3 μm) column was used by adding a mixture of 1% formic acid in acetonitrile/water (20 : 80, v/v, 350 μl/min). The total chromatographic run time was 5.5 min. The method was validated for the analysis of samples. The calibration curve was linear between 10 and 2000 ng/g for BH4 (r(2) = 0.995) , 10 and 5000 ng/g for DA (r(2) = 0.997) , 20 and 10000 ng/g for 5-HT (r(2) = 0.994) , NE (r(2) = 0.993) , and EP (r(2) = 0.993) , and 0.2 and 200 μg/g for Glu (r(2) = 0.996) and GABA (r(2) = 0.999) in the mouse brain tissues. As stated above, LC-MS/MS results were obtained and established to be a useful tool for the quantitative analysis of BH4, DA, 5-HT, NE, EP, Glu, and GABA in the experimental rodent brain. PMID:25258696

  7. Differentiation of benign and malignant focal liver lesions: value of virtual touch tissue quantification of acoustic radiation force impulse elastography.

    PubMed

    Guo, Le-Hang; Wang, Shu-Jun; Xu, Hui-Xiong; Sun, Li-Ping; Zhang, Yi-Feng; Xu, Jun-Mei; Wu, Jian; Fu, Hui-Jun; Xu, Xiao-Hong

    2015-03-01

    The purpose of this study was to investigate the value of virtual tissue quantification (VTQ) of acoustic radiation force impulse elastography for the differential diagnosis of benign and malignant focal liver lesions (FLLs). Thus, a total of 134 FLLs in 134 patients were included. VTQ measurement was performed for each lesion in which the shear wave velocity (SWV) was measured. The difference in SWV and SWV ratio of FLL to surrounding liver between malignant and benign FLLs was evaluated, and the cutoff value was investigated. Receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic performance. A total of 134 lesions including 55 (41.0%) malignant FLLs and 79 (59.0%) benign ones were analyzed. The SWV of malignant and benign FLLs was 2.95 ± 1.00 m/s and 1.69 ± 0.89 m/s, respectively. Significant difference in SWV was presented between malignant and benign FLLs (p < 0.001). The SWV ratio of each FLL to the surrounding liver parenchyma was 1.83 ± 1.32 for malignant and 1.26 ± 0.78 for benign FLLs (p < 0.001). The area under the ROC curve in distinguishing malignant from benign lesions was 0.824 for SWV and 0.660 for SWV ratio. The cutoff value for differential diagnosis was 2.13 m/s for SWV and 1.37 for SWV ratio. The associated sensitivity and specificity were 83.3 and 77.9% for SWV and 59.6 and 77.3% for SWV ratio, respectively. In conclusion, VTQ provides quantitative stiffness information of FLLs and is helpful in the differential diagnosis between malignant and benign FLLs, particularly for the patients who are not candidates for contrast-enhanced imaging such as CT, MRI or contrast-enhanced ultrasound. PMID:25691297

  8. 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

  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 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

  10. 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

  11. (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

  12. 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

  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. 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.

  15. Validation of Potential Reference Genes for qPCR in Maize across Abiotic Stresses, Hormone Treatments, and Tissue Types

    PubMed Central

    Lan, Hai; Gao, Shibin; Liu, Hailan; Liu, Jian; Cao, Moju; Pan, Guangtang; Rong, Tingzhao; Zhang, Suzhi

    2014-01-01

    The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α), tubulin beta (β-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types. PMID:24810581

  16. 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.

  17. Different extent of hypoxic-ischemic brain damage in newborn rats: histopathology, hemodynamic, virtual touch tissue quantification and neurobehavioral observation

    PubMed Central

    Wang, Si-Da; Liang, Shu-Yuan; Liao, Xin-Hong; Deng, Xiang-Fa; Chen, Yuan-Yuan; Liao, Chun-Yan; Wang, Lei; Tang, Shi; Li, Zhi-Xian

    2015-01-01

    Objective: To explore the correlation between pathological and ultrasound changes applying conventional ultrasound, Color Doppler ultrasound andVirtual Touch Tissue Quantification (VTQ) technique in newborn hypoxic-ischemic brain damage (HIBD) rat models. To provide theoretical basis for early diagnosis and treatment of HIBD neonatal. Methods: A total of 90 newborn Wistar rats were divided into ischemia, asphyxia and control group according to different HIBD molding methods. Conventional ultrasound, Color Doppler ultrasound and VTQ were applied on 3 h, 12 h, 24 h, 48 h and 72 h postoperative. After the observation of 72 h, 10 rats in each group were randomly selected for pathological specimens production. The rest rats were raised for 30 days for neuroethology detection. Results: In ischemia group and asphyxia group, there were 4 deaths and 6 deaths in the modeling process; the mortality rate was 13.33% (4/30) and 20.00% (6/30) respectively. For ischemia group, the systoli velocity (Vs), diastolic velocity (Vd) and resistance index (RI) of right middle cerebral artery (MCA) were significantly decreased after operation (P<0.05). For asphyxia group, the Vs and RI of right MCA were significantly decreased after operation (P<0.05), while the Vd of right MCA was significantly increased after operation (P<0.05), which lead to the postoperative RI value in each time point was all significantly lower than that in ischemia group (P<0.05). For ischemia group and asphyxia group, the VTQ results increased significantly postoperative (P<0.05), and compared with ischemia group and control group, the postoperative VTQ value in each time point was all significantly higher in asphyxia group (P<0.05). The neuroethology results were significantly lower in the ischemia group and asphyxia group (P<0.05), and the results in ischemia group were significantly higher than those of asphyxia group (P<0.05). And the results are consistent with the pathological findings. Conclusion: There is a

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

    PubMed

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

    2016-01-01

    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

  19. 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

  20. 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. PMID:27208623

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

    SciTech Connect

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

    1991-10-01

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

  2. Quantification of the Effect of Vertical Bone Resection of the Medial Proximal Tibia for Achieving Soft Tissue Balancing in Total Knee Arthroplasty

    PubMed Central

    Lee, Sung Hyun; Kang, Ho Won

    2016-01-01

    Background Degenerative osteoarthritis of the knee usually shows arthritic change in the medial tibiofemoral joint with severe varus deformity. In total knee arthroplasty (TKA), the medial release technique is often used for achieving mediolateral balancing. But, in a more severe varus knee, there are more difficult technical problems. Bony resection of the medial proximal tibia (MPT) as an alternative technique for achieving soft tissue balancing was assessed in terms of its effectiveness and possibility of quantification. Methods TKAs were performed in 78 knees (60 patients) with vertical bone resection of the MPT for soft tissue balancing from September 2011 to March 2013. During operation, the medial and lateral gaps were measured before and after the bony resection technique. First, the correlation between the measured thickness of the resected bone and the change in medial and lateral gaps was analyzed. Second, the possibility of quantification of each parameter was evaluated by linear regression and the coefficient ratio was obtained. Results A significant correlation was identified between alteration in the medial gap change in extension and the measured thickness of the vertically resected MPT (r = 0.695, p = 0.000). In the medial gap change in flexion, there was no statistical significance (r = 0.214, p = 0.059). When the MPT was resected at an average thickness of 8.25 ± 1.92 mm, the medial gap in extension was increased by 2.94 ± 0.87 mm. In simple linear regression, it was predictable that MPT resection at a thickness of 2.80 mm was required to increase the medial gap by 1.00 mm in knee extension. Conclusions The method of bone resection of the MPT can be considered effective with a predictable result for achieving soft tissue balancing in terms of quantification during TKA. PMID:26929799

  3. 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. PMID:25592328

  4. 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

  5. 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

  6. 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

  7. 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. PMID

  8. 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

  9. 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

  10. 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. PMID:26033549

  11. Wavefront aberration measurements and corrections through thick tissue using fluorescent microsphere reference beacons

    PubMed Central

    Azucena, Oscar; Crest, Justin; Cao, Jian; Sullivan, William; Kner, Peter; Gavel, Donald; Dillon, Daren; Olivier, Scot; Kubby, Joel

    2010-01-01

    We present a new method to directly measure and correct the aberrations introduced when imaging through thick biological tissue. A Shack-Hartmann wavefront sensor is used to directly measure the wavefront error induced by a Drosophila embryo. The wavefront measurements are taken by seeding the embryo with fluorescent microspheres used as “artificial guide-stars.” The wavefront error is corrected in ten millisecond steps by applying the inverse to the wavefront error on a micro-electro-mechanical deformable mirror in the image path of the microscope. The results show that this new approach is capable of improving the Strehl ratio by 2 times on average and as high as 10 times when imaging through 100 μm of tissue. The results also show that the isoplanatic half-width is approximately 19 μm resulting in a corrected field of view 38 μm in diameter around the guide-star. PMID:20721137

  12. Development of a novel DNA extraction method for identification and quantification of Mycobacterium avium subsp. paratuberculosis from tissue samples by real-time PCR.

    PubMed

    Park, Kun Taek; Allen, Andrew J; Davis, William C

    2014-04-01

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease in ruminants and possibly associated with human Crohn's disease. One impediment in furthering our understanding of this potential association has been the lack of an accurate method for detection of Map in affected tissues. Real time polymerase chain reaction (RT-PCR) methods have been reported to have different sensitivities in detection of Map. This is in part attributable to the difficulties of extracting Map DNA and removing PCR inhibitors from the clinical specimens. The maximum efficiency of RT-PCR can only be achieved by using high quality DNA samples. In this study, we present a novel pre-treatment method which significantly increases Map DNA recovery and decreases PCR inhibitors (p<0.05). When the pre-treatment method was combined with the DNeasy Blood and Tissue kit (Qiagen), PCR inhibition was not detected in any of three different RT-PCR methods tested in this study. The results obtained with the IS900 probe showed an excellent Kappa value (0.849) and a high correlation coefficient r (0.940) compared to the results of culture method. When used to examine unknown field samples (n=15), more positive tissues were identified with DNA extracts prepared with pre-treatment method than without (5 vs 3). This improved Map DNA extraction method from tissue samples will make RT-PCR a more powerful tool for a wide range of applications for Map identification and quantification. PMID:24534783

  13. High Resolution Systematic Digital Histological Quantification of Cardiac Fibrosis and Adipose Tissue in Phospholamban p.Arg14del Mutation Associated Cardiomyopathy

    PubMed Central

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

    2014-01-01

    Myocardial fibrosis can lead to heart failure and act as a substrate for cardiac arrhythmias. In dilated cardiomyopathy diffuse interstitial reactive fibrosis can be observed, whereas arrhythmogenic cardiomyopathy is characterized by fibrofatty replacement in predominantly the right ventricle. The p.Arg14del mutation in the phospholamban (PLN) gene has been associated with dilated cardiomyopathy and recently also with arrhythmogenic cardiomyopathy. Aim of the present study is to determine the exact pattern of fibrosis and fatty replacement in PLN p.Arg14del mutation positive patients, with a novel method for high resolution systematic digital histological quantification of fibrosis and fatty tissue in cardiac tissue. Transversal mid-ventricular slices (n = 8) from whole hearts were collected from patients with the PLN p.Arg14del mutation (age 48±16 years; 4 (50%) male). An in-house developed open source MATLAB script was used for digital analysis of Masson's trichrome stained slides (http://sourceforge.net/projects/fibroquant/). Slides were divided into trabecular, inner and outer compact myocardium. Per region the percentage of connective tissue, cardiomyocytes and fatty tissue was quantified. In PLN p.Arg14del mutation associated cardiomyopathy, myocardial fibrosis is predominantly present in the left posterolateral wall and to a lesser extent in the right ventricular wall, whereas fatty changes are more pronounced in the right ventricular wall. No difference in distribution pattern of fibrosis and adipocytes was observed between patients with a clinical predominantly dilated and arrhythmogenic cardiomyopathy phenotype. In the future, this novel method for quantifying fibrosis and fatty tissue can be used to assess cardiac fibrosis and fatty tissue in animal models and a broad range of human cardiomyopathies. PMID:24732829

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  15. Single-cell screening and quantification of transcripts in cancer tissues by second-harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Jing; Damayanti, Nur P.; Cho, Il-Hoon; Polar, Yesim; Badve, Sunil; Irudayaraj, Joseph M. K.

    2015-09-01

    Fluorescence-based single molecule techniques to interrogate gene expression in tissues present a very low signal-to-noise ratio due to the strong autofluorescence and other background signals from tissue sections. This report presents a background-free method using second-harmonic generation (SHG) nanocrystals as probes to quantify the messenger RNA (mRNA) of human epidermal growth receptor 2 (Her2) at single molecule resolution in specific phenotypes at single-cell resolution directly in tissues. Coherent SHG emission from individual barium titanium oxide (BTO) nanoprobes was demonstrated, allowing for a stable signal beyond the autofluorescence window. Her2 surface marker and Her2 mRNA were specifically labeled with BTO probes, and Her2 mRNA was quantified at single copy sensitivity in Her2 expressing phenotypes directly in cancer tissues. Our approach provides the first proof of concept of a cross-platform strategy to probe tissues at single-cell resolution in situ.

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

    PubMed

    Vancompernolle, Bram; Croes, Kim; Angenon, Geert

    2016-04-01

    Rapid, easy and reliable quantification of amino acids is crucial in research on plant amino acid metabolism and nutritional improvement of crops via enrichment of essential amino acids. A recently reported analysis method, based on solid phase extraction (SPE), derivatization with methyl chloroformate and gas chromatography-mass spectrometry was optimized and tested on three-week-old Arabidopsis thaliana leaf tissues. Optimization of the SPE cleanup yielded recovery rates of minimum 95% for all amino acids (except arginine). Variations in accuracy and precision did not exceed 12.5%, except for cysteine, histidine and tryptophane, which were excluded from analysis. Quantification of overlapping peaks for isoleucine/threonine and proline/asparagine was possible by selection of two specific fragment ions for each amino acid. Of the 16 selected amino acids, 14 were quantified successfully in at least 75% of the samples, while methionine and tyrosine were only quantifiable in 6% and 42%, respectively. A case study on the aspartate super pathway confirmed the applicability of the optimized method on wild type and genetically modified plants: external supplementation of methionine or lysine yielded a 146-fold or 27-fold increase in the respective absolute amino acid levels compared with the control treatment. Induced expression of dhdps-r1 (a mutated lysine biosynthesis gene encoding a feedback insensitive enzyme) caused an 83-fold increase in absolute lysine levels. PMID:26994331

  17. 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. PMID:23684523

  18. 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

  19. 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.

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

    PubMed

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

    2013-05-01

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

  1. [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

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

    SciTech Connect

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

    2011-11-15

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

  3. Single-cell screening and quantification of transcripts in cancer tissues by second-harmonic generation microscopy.

    PubMed

    Liu, Jing; Damayanti, Nur P; Cho, Il-Hoon; Polar, Yesim; Badve, Sunil; Irudayaraj, Joseph M K

    2015-09-01

    Fluorescence-based single molecule techniques to interrogate gene expression in tissues present a very low signal-to-noise ratio due to the strong autofluorescence and other background signals from tissue sections. This report presents a background-free method using second-harmonic generation (SHG) nanocrystals as probes to quantify the messenger RNA (mRNA) of human epidermal growth receptor 2 (Her2) at single molecule resolution in specific phenotypes at single-cell resolution directly in tissues. Coherent SHG emission from individual barium titanium oxide (BTO) nanoprobes was demonstrated, allowing for a stable signal beyond the autofluorescence window. Her2 surface marker and Her2 mRNA were specifically labeled with BTO probes, and Her2 mRNA was quantified at single copy sensitivity in Her2 expressing phenotypes directly in cancer tissues. Our approach provides the first proof of concept of a cross-platform strategy to probe tissues at single-cell resolution in situ. PMID:26405822

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

    PubMed

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

    2015-04-01

    The stability of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and chlorinated pesticides in frozen mussel tissue Standard Reference Materials (SRMs) stored at -80 °C was assessed by analyzing samples of SRM 1974, SRM 1974a, and SRM 1974b Organics in Mussel Tissue (Mytilus edulis) periodically over 25 y, 20 y, and 12 y, respectively. The most recent analyses were performed during the certification of the fourth release of this material, SRM 1974c. Results indicate the concentrations of these persistent organic pollutants have not changed during storage at -80 °C. In addition, brominated diphenyl ethers (BDEs) were quantified in each of the materials during this study. The stability information is important for on-going monitoring studies collecting large quantities of samples for future analyses (i.e., formally established specimen banking programs). Since all four mussel tissue SRMs were prepared from mussels collected at the same site in Dorchester Bay, MA, USA, the results provide a temporal trend study for these contaminants over a 17 year period (1987 to 2004). PMID:25711987

  5. Insulin receptor/IGF-I receptor hybrids are widely distributed in mammalian tissues: quantification of individual receptor species by selective immunoprecipitation and immunoblotting.

    PubMed

    Bailyes, E M; Navé, B T; Soos, M A; Orr, S R; Hayward, A C; Siddle, K

    1997-10-01

    The insulin receptor (IR) and type 1 insulin-like growth factor (IGF-I) receptor (IGFR) are both widely expressed in mammalian tissues, and are known to be capable of heteromeric assembly as insulin/IGF hybrid receptors, in addition to the classically described receptors. By selective immunoadsorption of radioligand/receptor complexes and by immunoblotting we have determined the fraction of insulin receptors and IGF receptors occurring as hybrids in different tissues. Microsomal membranes were isolated from tissue homogenates and solubilized with Triton X-100. Solubilized receptors were incubated with 125I-IGF-I, and radioligand/receptor complexes bound by IR-specific and IGFR-specific monoclonal antibodies were quantified. The fraction of IGF-I binding sites behaving as hybrids (anti-IR-bound/anti-IGFR-bound) was approx. 40% in liver and spleen, 70% in placenta, and 85-90% in skeletal muscle and heart, similar results being obtained in rabbit and human tissues. There was no correlation between the proportion of hybrids and the ratio of 125I-insulin/125I-IGF-I binding in different tissues. The fraction of 125I-insulin bound to hybrids was too low for accurate quantification, because of the relatively low affinity of hybrids for insulin. The fraction of insulin receptors present in hybrids was therefore determined by immunoblotting. Receptors in solubilized human placental microsomal membranes were precipitated with IR-specific or IGFR-specific monoclonal antibodies, and after SDS/PAGE, blots were prepared and probed with IR-specific and IGFR-specific antisera. It was found that 15% of IR and 80% of IGFR were present in hybrids. Consistent with these figures, the overall level of IR was estimated, by blotting with the respective antibodies at concentrations shown to give equal signals with equal amounts of receptor, to be 4-fold greater than IGFR. Overall it was concluded that a significant fraction of both IR and IGFR occurs as hybrids in most mammalian tissues

  6. 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.

  7. 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

  8. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  11. Dystrophin quantification

    PubMed Central

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

    2014-01-01

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

  12. 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

  13. Initial In Vivo Quantification of Tc-99m Sestamibi Uptake as a Function of Tissue Type in Healthy Breasts Using Dedicated Breast SPECT-CT

    PubMed Central

    Mann, Steve D.; Perez, Kristy L.; McCracken, Emily K. E.; Shah, Jainil P.; Wong, Terence Z.; Tornai, Martin P.

    2012-01-01

    A pilot study is underway to quantify in vivo the uptake and distribution of Tc-99m Sestamibi in subjects without previous history of breast cancer using a dedicated SPECT-CT breast imaging system. Subjects undergoing diagnostic parathyroid imaging studies were consented and imaged as part of this IRB-approved breast imaging study. For each of the seven subjects, one randomly selected breast was imaged prone-pendant using the dedicated, compact breast SPECT-CT system underneath the shielded patient support. Iteratively reconstructed and attenuation and/or scatter corrected images were coregistered; CT images were segmented into glandular and fatty tissue by three different methods; the average concentration of Sestamibi was determined from the SPECT data using the CT-based segmentation and previously established quantification techniques. Very minor differences between the segmentation methods were observed, and the results indicate an average image-based in vivo Sestamibi concentration of 0.10 ± 0.16 μCi/mL with no preferential uptake by glandular or fatty tissues. PMID:22956950

  14. Perihematomal Cerebral Tissue Iron Quantification on MRI Following Intracerebral Hemorrhage in Two Human Subjects: Proof of Principle.

    PubMed

    Chaudhary, Neeraj; Pandey, Aditya S; Merchak, Kevin; Gemmete, Joseph J; Chenevert, Tom; Xi, Guohua

    2016-01-01

    Spontaneous intracranial hemorrhage (ICH) is a common hemorrhagic stroke subtype with significant neurological sequelae. The management of ICH is usually supportive treatment in the neuro-intensive care setting, while the body humors deal with the hematoma. Treatment of the hematoma is usually expectant management unless there is neurological deterioration caused by mass effect from the hemorrhage. Some minimally invasive techniques have been explored for lysing and evacuating the hematoma, but none of them have gained a stronghold in the routine clinical management of this condition. Studies mainly in animal (rodent and porcine) ICH models have shown the role of bound and unbound iron in causing neurotoxicity following an ICH. There is currently no noninvasive method for assessing iron levels in the cerebral tissue following ICH. Our study intends to explore the role of magnetic resonance imaging (MRI) in establishing iron levels in cerebral tissue at the periphery of the hematoma following an ICH. Initially, an MRI phantom was constructed with varying concentrations of liquid iron preparation in a water bath container. Susceptibility weighted sequences were utilized to scan this phantom to generate T2* signal magnitude measurements corresponding to the iron concentration in the phantom. Encouraged by the reliability of the measurements on the phantom, patients with ICH were then recruited into this experimental study once the inclusion criteria were met. One control and two human subjects had their brains scanned in a 3 T MRI scanner utilizing the same susceptibility weighted sequence. We found that ICH perihematomal brain tissue iron susceptibility signal measurements were 4 times higher than those of the baseline control and normal contralateral brain tissue. Three different baseline measurements (one control and two contralateral normal brain) revealed a level of 0.1 mg/ml of iron concentration in the contralateral brain tissue in the identical anatomical

  15. 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. PMID:27095633

  16. 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

  17. RNA-Sequencing Quantification of Hepatic Ontogeny and Tissue Distribution of mRNAs of Phase II Enzymes in Mice

    PubMed Central

    Gunewardena, Sumedha; Cui, Julia Y.; Yoo, Byunggil; Zhong, Xiao-bo; Klaassen, Curtis D.

    2013-01-01

    Phase II conjugating enzymes play key roles in the metabolism of xenobiotics. In the present study, RNA sequencing was used to elucidate hepatic ontogeny and tissue distribution of mRNA expression of all major known Phase II enzymes, including enzymes involved in glucuronidation, sulfation, glutathione conjugation, acetylation, methylation, and amino acid conjugation, as well as enzymes for the synthesis of Phase II cosubstrates, in male C57BL/6J mice. Livers from male C57BL/6J mice were collected at 12 ages from prenatal to adulthood. Many of these Phase II enzymes were expressed at much higher levels in adult livers than in perinatal livers, such as Ugt1a6b, -2a3, -2b1, -2b5, -2b36, -3a1, and -3a2; Gsta1, -m1, -p1, -p2, and -z1; mGst1; Nat8; Comt; Nnmt; Baat; Ugdh; and Gclc. In contrast, hepatic mRNA expression of a few Phase II enzymes decreased during postnatal liver development, such as mGst2, mGst3, Gclm, and Mat2a. Hepatic expression of certain Phase II enzymes peaked during the adolescent stage, such as Ugt1a1, Sult1a1, Sult1c2, Sult1d1, Sult2as, Sult5a1, Tpmt, Glyat, Ugp2, and Mat1a. In adult mice, the total transcripts for Phase II enzymes were comparable in liver, kidney, and small intestine; however, individual Phase II enzymes displayed marked tissue specificity among the three organs. In conclusion, this study unveils for the first time developmental changes in mRNA abundance of all major known Phase II enzymes in mouse liver, as well as their tissue-specific expression in key drug-metabolizing organs. The age- and tissue-specific expression of Phase II enzymes indicate that the detoxification of xenobiotics is highly regulated by age and cell type. PMID:23382457

  18. 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.

  19. Quantification of phosphorus metabolites in human calf muscle and soft-tissue tumours from localized MR spectra acquired using surface coils

    NASA Astrophysics Data System (ADS)

    Doyle, V. L.; Payne, G. S.; Collins, D. J.; Verrill, M. W.; Leach, M. O.

    1997-04-01

    Metabolite concentrations determined from MR spectra provide more specific information than peak area ratios. This paper presents a method of quantification that allows metabolite concentrations to be determined from in vivo MR spectra acquired using a surface coil and ISIS localization. Corrections for the effects of field inhomogeneity produced by surface coils are based on a measured and calibrated spatial sensitivity field map for the coil. Account is taken of imperfections in pulse performance, coil loading effects and relaxation effects, the latter making use of published metabolite relaxation times. The technique is demonstrated on model solutions. The concentrations of the main metabolites in normal human calf muscle measured using this method are [PCr] = ; [Pi] = ; [NTP] = . Quantification of spectra acquired from soft-tissue tumours in patients both pre- and post-treatment showed that changes in metabolite concentrations are more sensitive to metabolic changes than changes in peak area ratios.

  20. 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

  1. Immunization of Wistar female rats with 255-Gy-irradiated Toxoplasma gondii: Tissue parasitic load and lactogenic quantification.

    PubMed

    Camossi, Lucilene Granuzzio; Fornazari, Felipe; Richini-Pereira, Virgínia Bodelão; Costa da Silva, Rodrigo; Cardia, Daniel Fontana Ferreira; Langoni, Helio

    2015-07-01

    Toxoplasma gondii is one of the most significant parasite, due its importance in veterinary medicine and in public health, considered a food-borne pathogens, there is no available drug treatments to eliminate it from animal tissue, this reinforce the search for a vaccine against this parasite. This study was aimed to evaluate the dynamic of the distribution of T. gondii in tissues of female Wistar rats and their milk, after the immunization by oral rote with irradiated tachyzoites. One week after pregnancy confirmation, rats was challenged by gavage with T. gondii bradyzoites, oocysts or tachyzoites of T. gondii. Forty-eight pregnant rats were grouped as follows: immunized and challenged with bradyzoites (BZ*); non-immunized and challenged with bradyzoites (BZ); immunized and challenged with oocysts (OC*); non-immunized and challenged with oocysts (OC); immunized and challenged with tachyzoites (TZ*); non-immunized and challenged with tachyzoites (TZ); only immunized (I); control group (C). After parturition, milk samples were collected for 3 weeks and then rats were sacrificed and the tissues and milk samples were researched for T. gondii parasite load determined by the quantitative PCR (qPCR). It was verified that the immunization with irradiated tachyzoites of T. gondii induced the reduction of parasitic load in muscle samples in rats challenged by bradyzoites and oocysts, although not enabled the development of sterile immunity. The detection of parasite DNA in milk was found throughout the lactation period, from immunized and non-immunized rats, however no differences were found in the parasite load caused by immunization. PMID:25936982

  2. Associative image analysis: a method for automated quantification of 3D multi-parameter images of brain tissue

    PubMed Central

    Bjornsson, Christopher S; Lin, Gang; Al-Kofahi, Yousef; Narayanaswamy, Arunachalam; Smith, Karen L; Shain, William; Roysam, Badrinath

    2009-01-01

    Brain structural complexity has confounded prior efforts to extract quantitative image-based measurements. We present a systematic ‘divide and conquer’ methodology for analyzing three-dimensional (3D) multi-parameter images of brain tissue to delineate and classify key structures, and compute quantitative associations among them. To demonstrate the method, thick (~100 μm) slices of rat brain tissue were labeled using 3 – 5 fluorescent signals, and imaged using spectral confocal microscopy and unmixing algorithms. Automated 3D segmentation and tracing algorithms were used to delineate cell nuclei, vasculature, and cell processes. From these segmentations, a set of 23 intrinsic and 8 associative image-based measurements was computed for each cell. These features were used to classify astrocytes, microglia, neurons, and endothelial cells. Associations among cells and between cells and vasculature were computed and represented as graphical networks to enable further analysis. The automated results were validated using a graphical interface that permits investigator inspection and corrective editing of each cell in 3D. Nuclear counting accuracy was >89%, and cell classification accuracy ranged from 81–92% depending on cell type. We present a software system named FARSIGHT implementing our methodology. Its output is a detailed XML file containing measurements that may be used for diverse quantitative hypothesis-driven and exploratory studies of the central nervous system. PMID:18294697

  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. Noninvasive Quantification of In Vitro Osteoblastic Differentiation in 3D Engineered Tissue Constructs Using Spectral Ultrasound Imaging

    PubMed Central

    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±2.4×104 at day 1 to 0.9±0.2×104 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. PMID:24465680

  5. 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. PMID:24465680

  6. 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

  7. 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

  8. Quantification of dexamethasone and corticosterone in rat biofluids and fetal tissue using highly sensitive analytical methods: assay validation and application to a pharmacokinetic study

    PubMed Central

    Samtani, Mahesh N.; Jusko, William J.

    2014-01-01

    A sensitive, specific, accurate and precise LC/MS/MS method was developed for the simultaneous measurement of dexamethasone and corticosterone in rat plasma. The method was extended to dexamethasone analysis in rat plasma ultrafiltrate and fetal tissues. Samples were processed using SPE involving Oasis HLB cartridges, which offered complete extraction recovery for the analytes. Samples were subsequently analyzed using LC/MS/MS. A structurally related corticosteroid, prednisolone, was used as the internal standard. Using a 500 μL plasma sample, limits of quantification of 0.2 and 2.0 ng/mL were achievable for dexamethasone and corticosterone. This level of sensitivity allowed characterization of maternal/fetal dexamethasone profiles after administration of multiple doses of dexamethasone sodium phosphate to rats. However, this sensitivity was not satisfactory for corticosterone during pharmacokinetic studies involving dexamethasone due to its strong adrenosuppressive effect. This led us to investigate the suitability of a commercially available radioimmunoassay kit, which through extensive testing and minor modifications was found to offer extremely sensitive, specific, accurate and precise analysis of corticosterone. Knowledge of the steroid profiles captured using these highly sensitive analytical tools may potentially help in the optimization of corticosteroid therapy during pregnancy. PMID:17385808

  9. Value of Virtual Touch Tissue Imaging Quantification for Evaluation of Ultrasound Breast Imaging-Reporting and Data System Category 4 Lesions.

    PubMed

    Li, Xiao-Long; Xu, Hui-Xiong; Bo, Xiao-Wan; Liu, Bo-Ji; Huang, Xian; Li, Dan-Dan; Guo, Le-Hang; Xu, Jun-Mei; Sun, Li-Ping; Fang, Lin; Xu, Xiao-Hong

    2016-09-01

    The purpose of the study was to evaluate the value of 2-D shear wave elastography (SWE) of virtual touch tissue imaging quantification (VTIQ) for ultrasound (US) Breast Imaging-Reporting and Data System (BI-RADS) category 4 lesions. One hundred sixteen lesions were subject to conventional US, conventional strain elastography (SE) of elasticity imaging (EI), acoustic radiation force impulse (ARFI)-induced SE of virtual touch tissue imaging (VTI) and VTIQ before biopsies. Of the 116 lesions, 69 (59.5%) were benign and 47 (40.5%) were malignant. Significant differences were found between benign and malignant lesions in EI score, VTI score and shear wave speed (SWS) on VTIQ (both p < 0.05). The cut-off values were EI score ≥4, VTI score ≥4 and SWS ≥3.49 m/s, respectively. The diagnostic performance of VTIQ in terms of area under receiver operating characteristic curve (AUROC) were the highest (i.e., AUROC = 0.907), in comparison with EI, VTI alone or a combination of both. The associated sensitivity, specificity and accuracy were 87.2%, 82.6% and 84.5%, respectively. The combination of VTI and VTIQ, however, was similar with US BI-RADS (p = 0.475) in sensitivity in that only two (4.3%) of 47 malignant lesions were misdiagnosed as benign that were BI-RADS category 4b on US. VTIQ is valuable to differentiate benign from malignant BI-RADS category 4 lesions, and the combination of VTI and VTIQ might be useful for patient selection before biopsy. PMID:27174418

  10. 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

  11. 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.

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

    PubMed

    Chase, Dorothy M; Elliott, Diane G; Pascho, Ronald J

    2006-07-01

    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. PMID:16921877

  13. Application of a probabilistic microstructural model to determine reference length and toe-to-linear region transition in fibrous connective tissue.

    PubMed

    Hurschler, Christof; Provenzano, Paolo P; Vanderby, Ray

    2003-06-01

    This study shows how a probabilistic microstructural model for fibrous connective tissue behavior can be used to objectively describe soft tissue low-load behavior. More specifically, methods to determine tissue reference length and the transition from the strain-stiffening "toe-region" to the more linear region of the stress-strain curve of fibrous connective tissues are presented. According to a microstructural model for uniaxially loaded collagenous tissues, increasingly more fibers are recruited and bear load with increased tissue elongation. Fiber recruitment is represented statistically according to a Weibull probability density function (PDF). The Weibull PDF location parameter in this formulation corresponds to the stretch at which the first fibers begin to bear load and provides a convenient method of determining reference length. The toe-to-linear region transition is defined by utilizing the Weibull cumulative distribution function (CDF) which relates the fraction of loaded fibers to the tissue elongation. These techniques are illustrated using representative tendon and ligament data from the literature, and are shown to be applicable retrospectively to data from specimens that are not heavily preloaded. The reference length resulting from this technique provides an objective datum from which to calculate stretch, strain, and tangent modulus, while the Weibull CDF provides an objective parameter with which to characterize the limits of low-load behavior. PMID:12929247

  14. Development and validation of HPLC method with fluorometric detection for quantification of bisnaphthalimidopropyldiaminooctane in animal tissues following administration in polymeric nanoparticles.

    PubMed

    Segundo, Marcela A; Abreu, Vera L R G; Osório, Marcelo V; Nogueira, Sonia; Lin, Paul Kong Thoo; Cordeiro-da-Silva, Anabela; Lima, Sofia A C

    2016-02-20

    A simple, sensitive and specific high-performance liquid chromatography method for the quantification of bisnaphthalimidopropyldiaminooctane (BNIPDaoct), a potent anti-Leishmania compound, incorporated into poly(d,l-lactide-co-glycolic acid) (PLGA) nanoparticles was developed and validated toward bioanalysis application. Biological tissue extracts were injected into a reversed-phase monolithic column coupled to a fluorimetric detector (λexc=234nm, λem=394nm), using isocratic elution with aqueous buffer (acetic acid/acetate 0.10M, pH 4.5, 0.010M octanesulfonic acid) and acetonitrile, 60:40 (v/v) at a flow rate of 1.5mLmin(-1). The run time was 6min, with a BNIPDaoct retention time of 3.3min. Calibration curves were linear for BNIPDaoct concentrations ranging from 0.002 to 0.100μM. Matrix effects were observed and calibration curves were performed using the different organ (spleen, liver, kidney, heart and lung) extracts. The method was found to be specific, accurate (97.3-106.8% of nominal values) and precise for intra-day (RSD<1.9%) and inter-day assays (RSD<7.2%) in all matrices. Stability studies showed that BNIPDaoct was stable in all matrices after standing for 24h at room temperature (20°C) or in the autosampler, and after three freeze-thaw cycles. Mean recoveries of BNIPDaoct spiked in mice organs were >88.4%. The LOD and LOQ for biological matrices were ≤0.8 and ≤1.8nM, respectively, corresponding to values ≤4 and ≤9nmolg(-1) in mice organs. The method developed was successfully applied to biodistribution assessment following intravenous administration of BNIPDaoct in solution or incorporated in PLGA nanoparticles. PMID:26765266

  15. Preliminary study on the role of virtual touch tissue quantification combined with a urinary β2-microglobulin test on the early diagnosis of gouty kidney damage.

    PubMed

    Tian, Fei; Wang, Zheng-Bin; Meng, Dong-Mei; Liu, Rong-Gui; Zhang, Hai-Yan; Li, Hui-Ying; Lv, Fei-Fei

    2014-07-01

    The goal of the work described here was to evaluate the role of virtual touch tissue quantification (VTQ) combined with urinary β2-microglobulin (β2-MG) measurement in the early diagnosis of gouty kidney damage. Two hundred fifty-nine patients with gouty kidney damage and 200 healthy control subjects were tested. The shear wave velocity (SWV) of the renal parenchyma and sinus as determined with VTQ and the urinary β2-MG level of the two groups were analyzed. Although there were no significant differences in age, body mass index, creatinine level and blood urea nitrogen between the two groups (all p's > 0.05), the aforementioned parameters were higher in the group with gouty kidney damage than in the control group. Urinary β2-MG levels of the patients with kidney damage were significantly higher than those of the control subjects (t = 6.38, p < 0.01). The SWV of the renal parenchyma was higher than that of the sinus in both groups. Compared with controls, patients with kidney damage had significantly increased renal parenchyma and sinus SWVs (all p-values < 0.05). Urinary β2-MG level was positively linearly correlated with the SWV of renal parenchyma in patients with kidney damage (r = 0.442, p < 0.0001). However, there was no correlation between urinary β2-MG level and the SWV of the sinus in patients with kidney damage (r = 0). In the control group, there was no correlation between urinary β2-MG level and the SWV of the renal parenchyma or sinus. The elasticity of the kidney as determined with VTQ, combined with the urinary β2-MG level, may be helpful in the early diagnosis of gouty kidney damage. PMID:24642221

  16. 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

  17. 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

  18. 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

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

    NASA Astrophysics Data System (ADS)

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

    2009-01-01

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

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

    PubMed Central

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

    2015-01-01

    In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or ‘housekeeping’) gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a, Rps18, Rps29, Sdha, Tbp and Ubc) across several juvenile and adult rat tissues (liver, adrenal, prostate, fat pad, testis and ovaries), both under normal conditions and following exposure to various chemicals during development. Employing NormFinder and BestKeeper softwares, we found Hprt and Sdha to be amongst the most stable genes across normal and manipulated tissues, with several others also being suitable for most tissues. Tbp and B2m displayed highest variability in transcript levels between tissues and developmental stages. It was also observed that the reference genes were most unstable in liver and testis following toxicological exposure. For future studies, we propose the use of more than one verified reference gene and the continuous monitoring of their suitability under various experimental conditions, including toxicological studies, based on changes in threshold (Ct) values from cDNA samples having been reverse-transcribed from a constant input concentration of RNA. PMID:25825680

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

    PubMed

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

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene. PMID:24776823

  2. 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

  3. Vanadium poisoning of cattle with basic slag. Concentrations in tissues from poisoned animals and from a reference, slaughterhouse material.

    PubMed

    Frank, A; Madej, A; Galgan, V; Petersson, L R

    1996-03-01

    In northern Sweden, 23 heifers out of 98 cattle died of acute vanadium toxicity in a 10-day period. Eight months earlier a pasture had been fertilized with basic slag, containing 3% vanadium. The fertilizer was laid on the surface without being ploughed in. Mainly heifers, and some cows, were fed with basic slag-contaminated fresh hay. The first signs of illness appeared 11 days later, and the first case of death appeared 14 days after the initial clinical signs. The signs were diffuse and difficult to interpret. Inappetence, black diarrhea, lethargy, dehydration and spontaneous abortions occurred. Further, pulmonary lesions, conjunctivitis, neurological disturbances such as depression, leg incoordination, and paralysis of the hind limbs and face were also noted. Although feeding with the contaminated hay was stopped at the outbreak of the toxicity, the 23 animals died or had to be slaughtered, and at necropsy of another heifer 4 weeks later, large amounts of basic slag were still found in the alimentary tract. High vanadium concentrations were found in the liver, kidneys, spleen and urine, 5.9, 5.5, 1.9 and 4.8 mg/kg w.w., respectively. In bone tissue (coccygeal vertebrae), the highest value in an acutely poisoned heifer was 0.680 mg/kd d.w., in the same range as that of an experimentally poisoned sheep. Surviving heifers were more affected than cows; the state of health of these heifers gradually deteriorated and, therefore, a few were slaughtered 3 months later. Because of residual neurological disturbances and decreased milk production, the rest of the herd was slaughtered 5 months after the outbreak, and samples were collected and analyzed. Elevated vanadium concentrations were found in the organs, especially in the spleen, where values of 1.40 and 1.42 mg/kg w.w. were found in 2 heifers at 3 months. The values in heifers (n = 6) were decreased 5 months after the outbreak. The median concentrations were somewhat higher in the liver than in the spleen, and

  4. 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. PMID:26275489

  5. 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.

  6. 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

  7. Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction.

    PubMed

    Zornhagen, K W; Kristensen, A T; Hansen, A E; Oxboel, J; Kjaer, A

    2015-12-01

    Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. β-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set-up. PMID:24995963

  8. 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

  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. PMID:27621722

  10. 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

  11. 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

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

    PubMed

    Peng, Xinjian; McCormick, David L

    2016-08-01

    Oral squamous cell carcinomas (OSCCs) induced in F344 rats by 4-nitroquinoline-1-oxide (4-NQO) demonstrate considerable phenotypic similarity to human oral cancers and the model has been widely used for carcinogenesis and chemoprevention studies. Molecular characterization of this model needs reliable reference genes (RGs) to avoid false- positive and -negative results for proper interpretation of gene expression data between tumor and adjacent normal tissues. Microarray analysis of 11 pairs of OSCC and site-matched phenotypically normal oral tissues from 4-NQO-treated rats identified 10 stably expressed genes in OSCC compared to adjacent normal tissues (p>0.5, CV<15%) that could serve as potential RGs in this model. The commonly used 27 RGs in the rat were also analyzed based on microarray data and most of them were found unsuitable for RGs in this model. Traditional RGs such as ACTB and GAPDH were significantly altered in OSCC compared to adjacent normal tissues (p<0.01, n=11); however, the Hsp90ab1 was ranked as the best RG candidate and the combination of Hsp90ab1 and HPRT1 was identified by NormFinder to be a superior reference for gene normalization among the commonly used RGs. This result was also validated by RT-PCR based on the selected top RG candidate pool. These data suggest that there are no common RGs suitable for different models and RG(s) should be identified before gene expression analysis. We successfully identified Hsp90ab1 as a stable RG in 4-NQO-induced OSCC compared to adjacent normal tissues in F344 rats. The combination of two stably expressed genes may be a better option for gene normalization in tissue samples. PMID:27375172

  13. Seasonal changes in tissue weight and biochemical composition of the bivalve nucula turgida in Dublin Bay with reference to gametogenesis

    NASA Astrophysics Data System (ADS)

    Davis, J. P.; Wilson, J. G.

    The biochemical composition and tissue weight of Nucula turgida were monitored over 18 months and are reported for a standard animal of 8 mm shell length. The tissue flesh dry weight was found to increase steadily during the spring. After reaching a maximum value of 11.0 mg in July, it decreased until the end of winter when the weight was 5.5 mg. The protein and lipid content also reached a maximum in July but the carbohydrate content continued to increase for another month. The sudden decrease in all the biochemical components and the tissue weight during September coincides with the main period of spawning. A difference in biochemical composition between the sexes was noted in pre-spawning months (June to early September). Females showed an increase in the amount of lipid while males had a larger proportion of protein; no differences were apparent in the carbohydrate content. The seasonal changes are compared with those reported for other bivalves.

  14. The isoenzymes of carbonic anhydrase: tissue, subcellular distribution and functional significance, with particular reference to the intestinal tract

    PubMed Central

    Carter, M. J.; Parsons, D. S.

    1971-01-01

    1. The total carbonic anhydrase activity in some guinea-pig tissues has been measured using a pH-stat procedure. Stomach, gall bladder, proximal colon and caecum all possess more carbonic anhydrase activity per unit amount of protein than does whole blood. 2. The carbonic anhydrase activity of the small intestine is low. Reasons are given for supposing that activity found there is not entirely due to contamination by whole blood, and it is suggested that in this tissue the enzyme may be localized in some cell type other than the columnar absorbing cells. 3. Evidence is presented which indicates that heavy metals interfere with the activity of the enzyme as measured in tissue homogenates. 4. The distribution and concentration of the two major isoenzymes of carbonic anhydrase have been measured in different tissues. Blood and proximal colon contain both isoenzymes in comparable concentrations, the ratio of the concentration of the `low activity' isoenzyme to that of the `high activity' being about 2. The gastric mucosa contains much `high activity' carbonic anhydrase, but only a negligible amount of the `low activity' isoenzyme. In the caecal mucosa, the `low activity' isoenzyme is predominant, the ratio of its concentration to that of the `high activity' isoenzyme being about 9. It is also found that more than 1·5% of the protein in the caecal mucosa is accounted for as carbonic anhydrase enzymes. 5. It is found that some 45% of the total carbonic anhydrase activity of sucrose homogenates of the guinea-pig colon is bound to particles. The activity is located mainly in the nuclear and microvillous fraction and in the `high-speed supernatant' fraction. The form of enzyme bound is largely of the `high activity' variety. When the tissue is homogenized in potassium chloride solutions less than 4% of the total activity is recovered in particulate fractions. The amount of activity which is bound to particulate fractions increases as the ionic strength or pH of the

  15. 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

  16. Validation of adequate endogenous reference genes for reverse transcription-qPCR studies in human post-mortem brain tissue of SIDS cases.

    PubMed

    El-Kashef, Noha; Gomes, Iva; Mercer-Chalmers-Bender, Katja; Schneider, Peter M; Rothschild, Markus A; Juebner, Martin

    2015-12-01

    Sudden infant death syndrome (SIDS) is the main cause of post-neonatal infant death in most developed countries. It is still of ambiguous etiology. Gene expression studies of relevant target genes using reverse transcription quantitative real-time PCR (RT-qPCR) in SIDS cases, and comparing them with age-matched controls, could help in understanding the pathogenesis of SIDS. However, selecting inadequate reference genes used for normalization of the RT-qPCR gene expression data can give misleading results. The aim of the present study was to identify reference genes with the most stable expression in post-mortem brainstem samples of SIDS and control cases. Among the five candidate reference genes (GAPDH, GUSB, HMBS, SDHA, UBXN6) studied in both groups, SDHA and UBXN6 were identified as the most stable. To further demonstrate the importance of using validated genes for RT-qPCR data normalization, the expression of a potential gene of interest in SIDS, the RPS27A gene, was evaluated using validated versus non-validated reference genes for normalization. This gene encodes the ubiquitin protein that has been shown in other pathological studies to be induced in SIDS. Using the identified most stable genes for normalization of RPS27A gene expression data revealed, as expected, a statistically significant up-regulation in SIDS as compared to the controls. However, using a single unstable reference gene for normalization resulted in no significant differences in transcript abundance of RPS27A between SIDS and the controls. This emphasizes the need for validation of the suitability of reference genes used in a given tissue type under certain experimental conditions. PMID:26434654

  17. Validation of the reference tissue model for estimation of dopaminergic D2-like receptor binding with [18F](N-methyl)benperidol in humans.

    PubMed

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

    2008-04-01

    Positron emission tomography measurements of dopaminergic D2-like receptors may provide important insights into disorders such as Parkinson's disease, schizophrenia, dystonia and Tourette's syndrome. The positron emission tomography (PET) radioligand [18F](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 the 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<.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

  18. Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data

    PubMed Central

    2013-01-01

    Background Data normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. However, the transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the performance of the target gene(s). This issue has been scarcely approached in woody species such as grapevine. Results A statistical criterion was applied to select a sub-set of 19 candidate reference genes from a total of 242 non-differentially expressed (NDE) genes derived from a RNA-Seq experiment comprising ca. 500 million reads obtained from 14 table-grape genotypes sampled at four phenological stages. From the 19 candidate reference genes, VvAIG1 (AvrRpt2-induced gene) and VvTCPB (T-complex 1 beta-like protein) were found to be the most stable ones after comparing the complete set of genotypes and phenological stages studied. This result was further validated by qPCR and geNorm analyses. Conclusions Based on the evidence presented in this work, we propose to use the grapevine genes VvAIG1 or VvTCPB or both as a reference tool to normalize RNA expression in qPCR assays or other quantitative method intended to measure gene expression in berries and other tissues of this fruit crop, sampled at different developmental stages and physiological conditions. PMID:24330674

  19. 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. PMID:26138891

  20. 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

  1. 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

  2. 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

  3. 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

  4. Arterial input functions (AIFs) measured directly from arteries with low and standard doses of contrast agent, and AIFs derived from reference tissues

    PubMed Central

    Wang, Shiyang; Fan, Xiaobing; Medved, Milica; Pineda, Federico D.; Yousuf, Ambereen; Oto, Aytekin; Karczmar, Gregory S.

    2016-01-01

    Measurements of arterial input function (AIF) can have large systematic errors at standard contrast agent doses in dynamic contrast enhanced MRI (DCE-MRI). We compared measured AIFs from low dose (AIFLD) and standard dose (AIFSD) contrast agent injections, as well as the AIF derived from a muscle reference tissue and artery (AIFref). Twenty-two prostate cancer patients underwent DCE-MRI. Data were acquired on a 3 T scanner using an mDixon sequence. Gadobenate dimeglumine was injected twice, at doses of 0.015 and 0.085 mmol/kg. Directly measured AIFs were fitted with empirical mathematical models (EMMs) and compared to the AIF derived from a muscle reference tissue (AIFref). EMMs accurately fitted the AIFs. The 1st and 2nd pass peaks were visualized in AIFLD, but not in AIFSD, thus the peak and shape of AIFSD could not be accurately measured directly. The average scaling factor between AIFSD and AIFLD in the washout phase was only 56% of the contrast dose ratio (~6:1). The shape and magnitude of AIFref closely approximated that of AIFLD after empirically determined dose-dependent normalization. This suggests that AIFref may be a good approximation of the local AIF. PMID:26523650

  5. 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

  6. Development and validation of a LC-MS assay for the quantification of ikh12 a novel anti-tumor candidate in rat plasma and tissues and its application in a pharmacokinetic study.

    PubMed

    Otaegui, Dorleta; Masdeu, Carme; Aldaba, Eneko; Vara, Yosu; Zubia, Aizpea; San Sebastian, Eider; Alcalá, Maria; Villafruela, Sergio; Cossío, Fernando P; Rodriguez-Gascón, Alicia

    2015-08-01

    IKH12 is a novel histone deacetylase 6 selective inhibitor. A rapid and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of IKH12 in rat plasma and tissue with kendine 91 as internal standard (IS). The samples were prepared by liquid-liquid extraction with tert-butyl methyl ether. The chromatographic separation was accomplished by using a Zorbax Extend C18 4.6 × 150 mm, 5 µm column, with a mobile phase consisting of methanol and 0.1% formic acid (75:25 v/v). Multiple reaction monitoring, using electrospray ionization in positive ion mode, was employed to quantitatively detect IKH12 and IS. The monitored transitions were set at m/z 418 → 252 and 444 → 169 for IKH12 and kendine 91, respectively. The calibration curve was linear over the concentration range 2-1000 ng mL(-1) . The intra- and inter-assay precision and accuracy of the quality controls and the limit of quantification were satisfactory in all cases (according to European Medicines Agency guidelines). Stability studies showed that plasma samples were stable in the chromatography rack for 24 h and at -80°C for 2 months and also after three freeze-thaw cycles. This method was successfully applied to a pharmacokinetic study of IKH12 in rat. PMID:25616154

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

    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.

  8. 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)

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

    PubMed Central

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

    2015-01-01

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

  10. 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