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.

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

PubMed

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

2016-01-01

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

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

Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues

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

Nie, Song; Shi, Tujin; Fillmore, Thomas L.

Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low ng/mL to sub-ng/mL level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundant but biologically important proteins (e.g., ≤100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging. To address this need, we have developed an antibody-independent Deep-Dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide enrichment combined withmore » precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ~5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue has been demonstrated to enable precise quantification of endogenous proteins at ~10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibody is not available.« less

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.

Histological quantification of brain tissue inflammatory cell infiltration after focal cerebral infarction: a systematic review.

PubMed

Russek, Natanya S; Jensen, Matthew B

2014-03-01

Ischemic stroke is a leading cause of death and disability, and current treatments to limit tissue injury and improve recovery are limited. Cerebral infarction is accompanied by intense brain tissue inflammation involving many inflammatory cell types that may cause both negative and positive effects on outcomes. Many potential neuroprotective and neurorestorative treatments may affect, and be affected by, this inflammatory cell infiltration, so that accurate quantification of this tissue response is needed. We performed a systematic review of histological methods to quantify brain tissue inflammatory cell infiltration after cerebral infarction. We found reports of multiple techniques to quantify different inflammatory cell types. We found no direct comparison studies and conclude that more research is needed to optimize the assessment of this important stroke outcome.

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

PubMed

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

2015-01-01

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. 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. miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that. 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.

Optimized approaches for quantification of drug transporters in tissues and cells by MRM proteomics.

PubMed

Prasad, Bhagwat; Unadkat, Jashvant D

2014-07-01

Drug transporter expression in tissues (in vivo) usually differs from that in cell lines used to measure transporter activity (in vitro). Therefore, quantification of transporter expression in tissues and cell lines is important to develop scaling factor for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated drug disposition. Since traditional immunoquantification methods are semiquantitative, targeted proteomics is now emerging as a superior method to quantify proteins, including membrane transporters. This superiority is derived from the selectivity, precision, accuracy, and speed of analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, LC-MS/MS proteomics has broader applicability because it does not require selective antibodies for individual proteins. There are a number of recent research and review papers that discuss the use of LC-MS/MS for transporter quantification. Here, we have compiled from the literature various elements of MRM proteomics to provide a comprehensive systematic strategy to quantify drug transporters. This review emphasizes practical aspects and challenges in surrogate peptide selection, peptide qualification, peptide synthesis and characterization, membrane protein isolation, protein digestion, sample preparation, LC-MS/MS parameter optimization, method validation, and sample analysis. In particular, bioinformatic tools used in method development and sample analysis are discussed in detail. Various pre-analytical and analytical sources of variability that should be considered during transporter quantification are highlighted. All these steps are illustrated using P-glycoprotein (P-gp) as a case example. Greater use of quantitative transporter proteomics will lead to a better understanding of the role of drug transporters in drug disposition.

Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non-Small Cell Lung Cancer Tissue.

PubMed

Kristof, Jessica; Sakrison, Kellen; Jin, Xiaoping; Nakamaru, Kenji; Schneider, Matthias; Beckman, Robert A; Freeman, Daniel; Spittle, Cindy; Feng, Wenqin

2017-01-01

In preclinical studies, heregulin ( HRG ) expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti-epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non-small cell lung cancer (NSCLC), a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG -positive and HRG -negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping) genes ( HMBS, IPO8 , and EIF2B1 ), which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

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.

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.

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

PubMed

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

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. 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 to discriminate

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

Quantification of HCV RNA in Liver Tissue by bDNA Assay.

PubMed

Dailey, P J; Collins, M L; Urdea, M S; Wilber, J C

1999-01-01

With this statement, Sherlock and Dooley have described two of the three major challenges involved in quantitatively measuring any analyte in tissue samples: the distribution of the analyte in the tissue; and the standard of reference, or denominator, with which to make comparisons between tissue samples. The third challenge for quantitative measurement of an analyte in tissue is to ensure reproducible and quantitative recovery of the analyte on extraction from tissue samples. This chapter describes a method that can be used to measure HCV RNA quantitatively in liver biopsy and tissue samples using the bDNA assay. All three of these challenges-distribution, denominator, and recovery-apply to the measurement of HCV RNA in liver biopsies.

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.

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

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.

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

PubMed

Oberbach, Andreas; Schlichting, Nadine; Neuhaus, Jochen; Kullnick, Yvonne; Lehmann, Stefanie; Heinrich, Marco; Dietrich, Arne; Mohr, Friedrich Wilhelm; von Bergen, Martin; Baumann, Sven

2014-12-05

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a

An approach for quantification of platinum distribution in tissues by LA-ICP-MS imaging using isotope dilution analysis.

PubMed

Moraleja, I; Mena, M L; Lázaro, A; Neumann, B; Tejedor, A; Jakubowski, N; Gómez-Gómez, M M; Esteban-Fernández, D

2018-02-01

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been revealed as a convenient technique for trace elemental imaging in tissue sections, providing elemental 2D distribution at a quantitative level. For quantification purposes, in the last years several approaches have been proposed in the literature such as the use of CRMs or matrix matched standards. The use of Isotope Dilution (ID) for quantification by LA-ICP-MS has been also described, being mainly useful for bulk analysis but not feasible for spatial measurements so far. In this work, a quantification method based on ID analysis was developed by printing isotope-enriched inks onto kidney slices from rats treated with antitumoral Pt-based drugs using a commercial ink-jet device, in order to perform an elemental quantification in different areas from bio-images. For the ID experiments 194 Pt enriched platinum was used. The methodology was validated by deposition of natural Pt standard droplets with a known amount of Pt onto the surface of a control tissue, where could be quantified even 50pg of Pt, with recoveries higher than 90%. The amount of Pt present in the whole kidney slices was quantified for cisplatin, carboplatin and oxaliplatin-treated rats. The results obtained were in accordance with those previously reported. The amount of Pt distributed between the medullar and cortical areas was also quantified, observing different behavior for the three drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

Lao, Zhiqiang; Zheng, Xin

2011-03-01

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

Quantification of the xenoestrogens 4-tert.-octylphenol and bisphenol A in water and in fish tissue based on microwave assisted extraction, solid-phase extraction and liquid chromatography-mass spectrometry.

PubMed

Pedersen, S N; Lindholst, C

1999-12-09

Extraction methods were developed for quantification of the xenoestrogens 4-tert.-octylphenol (tOP) and bisphenol A (BPA) in water and in liver and muscle tissue from the rainbow trout (Oncorhynchus mykiss). The extraction of tOP and BPA from tissue samples was carried out using microwave-assisted solvent extraction (MASE) followed by solid-phase extraction (SPE). Water samples were extracted using only SPE. For the quantification of tOP and BPA, liquid chromatography mass spectrometry (LC-MS) equipped with an atmospheric pressure chemical ionisation interface (APCI) was applied. The combined methods for tissue extraction allow the use of small sample amounts of liver or muscle (typically 1 g), low volumes of solvent (20 ml), and short extraction times (25 min). Limits of quantification of tOP in tissue samples were found to be approximately 10 ng/g in muscle and 50 ng/g in liver (both based on 1 g of fresh tissue). The corresponding values for BPA were approximately 50 ng/g in both muscle and liver tissue. In water, the limit of quantification for tOP and BPA was approximately 0.1 microg/l (based on 100 ml sample size).

Reference Models for Multi-Layer Tissue Structures

DTIC Science & Technology

2016-09-01

simulation,  finite   element  analysis 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC...Physiologically realistic, fully specimen-specific, nonlinear reference models. Tasks. Finite element analysis of non-linear mechanics of cadaver...models. Tasks. Finite element analysis of non-linear mechanics of multi-layer tissue regions of human subjects. Deliverables. Partially subject- and

Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications

PubMed Central

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

2016-01-01

Quantifying confocal images to enable location of specific proteins of interest in three-dimensional (3D) is important for many tissue engineering (TE) applications. Quantification of protein localization is essential for evaluation of specific scaffold constructs for cell growth and differentiation for application in TE and tissue regeneration strategies. Although obtaining information regarding protein expression levels is important, the location of proteins within cells grown on scaffolds is often the key to evaluating scaffold efficacy. Functional epithelial cell monolayers must be organized with apicobasal polarity with proteins specifically localized to the apical or basolateral regions of cells in many organs. In this work, a customized program was developed using the LabVIEW platform to quantify protein positions in Z-stacks of confocal images of epithelial cell monolayers. The program's functionality is demonstrated through salivary gland TE, since functional salivary epithelial cells must correctly orient many proteins on the apical and basolateral membranes. Bio-LabVIEW Image Matrix Evaluation (Bio-LIME) takes 3D information collected from confocal Z-stack images and processes the fluorescence at each pixel to determine cell heights, nuclei heights, nuclei widths, protein localization, and cell count. As a demonstration of its utility, Bio-LIME was used to quantify the 3D location of the Zonula occludens-1 protein contained within tight junctions and its change in 3D position in response to chemical modification of the scaffold with laminin. Additionally, Bio-LIME was used to demonstrate that there is no advantage of sub-100 nm poly lactic-co-glycolic acid nanofibers over 250 nm fibers for epithelial apicobasal polarization. Bio-LIME will be broadly applicable for quantification of proteins in 3D that are grown in many different contexts. PMID:27758134

Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications.

PubMed

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

2016-11-01

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

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

Refer prior to biopsy of suspected appendicular soft tissue sarcoma.

PubMed

Elliott, Robert S J; Flint, Michael; French, Gary

2012-11-23

Appendicular soft tissue tumours are rare and inappropriate investigation can result in unnecessary loss of limb or life. We reviewed the investigation and referrals of patients to our institution. This is a review of prospectively collected data stored in a tumour registry database. We included all patients (126) referred to the service for investigation and management with a primary soft tissue tumour in 2006 and 2007. There was a highly significant association (RR=6.2) between pre referral procedures (PRPs) and suffering a complication (P<0.0001) in comparison to non-biopsied referrals (NBRs). Those referred by general surgeons were more likely (RR=2.6) to have undergone PRP (p<0.0017). The median interval between referral and senior author review was 8 days for the PRP group and 10 days for the NBR group (P=0.2574). Biopsy of suspected appendicular soft tissue sarcoma should be performed by a tumour specialist or in prior consultation with, to minimise adverse outcomes. There was minimal delay till review by an orthopaedic tumour specialist at Middlemore Hospital and achieving a tissue diagnosis does not expedite this.

Multi-tissue partial volume quantification in multi-contrast MRI using an optimised spectral unmixing approach.

PubMed

Collewet, Guylaine; Moussaoui, Saïd; Deligny, Cécile; Lucas, Tiphaine; Idier, Jérôme

2018-06-01

Multi-tissue partial volume estimation in MRI images is investigated with a viewpoint related to spectral unmixing as used in hyperspectral imaging. The main contribution of this paper is twofold. It firstly proposes a theoretical analysis of the statistical optimality conditions of the proportion estimation problem, which in the context of multi-contrast MRI data acquisition allows to appropriately set the imaging sequence parameters. Secondly, an efficient proportion quantification algorithm based on the minimisation of a penalised least-square criterion incorporating a regularity constraint on the spatial distribution of the proportions is proposed. Furthermore, the resulting developments are discussed using empirical simulations. The practical usefulness of the spectral unmixing approach for partial volume quantification in MRI is illustrated through an application to food analysis on the proving of a Danish pastry. Copyright © 2018 Elsevier Inc. All rights reserved.

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

PubMed

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

2013-12-03

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

Blood pool and tissue phase patient motion effects on 82rubidium PET myocardial blood flow quantification.

PubMed

Lee, Benjamin C; Moody, Jonathan B; Poitrasson-Rivière, Alexis; Melvin, Amanda C; Weinberg, Richard L; Corbett, James R; Ficaro, Edward P; Murthy, Venkatesh L

2018-03-23

Patient motion can lead to misalignment of left ventricular volumes of interest and subsequently inaccurate quantification of myocardial blood flow (MBF) and flow reserve (MFR) from dynamic PET myocardial perfusion images. We aimed to identify the prevalence of patient motion in both blood and tissue phases and analyze the effects of this motion on MBF and MFR estimates. We selected 225 consecutive patients that underwent dynamic stress/rest rubidium-82 chloride ( 82 Rb) PET imaging. Dynamic image series were iteratively reconstructed with 5- to 10-second frame durations over the first 2 minutes for the blood phase and 10 to 80 seconds for the tissue phase. Motion shifts were assessed by 3 physician readers from the dynamic series and analyzed for frequency, magnitude, time, and direction of motion. The effects of this motion isolated in time, direction, and magnitude on global and regional MBF and MFR estimates were evaluated. Flow estimates derived from the motion corrected images were used as the error references. Mild to moderate motion (5-15 mm) was most prominent in the blood phase in 63% and 44% of the stress and rest studies, respectively. This motion was observed with frequencies of 75% in the septal and inferior directions for stress and 44% in the septal direction for rest. Images with blood phase isolated motion had mean global MBF and MFR errors of 2%-5%. Isolating blood phase motion in the inferior direction resulted in mean MBF and MFR errors of 29%-44% in the RCA territory. Flow errors due to tissue phase isolated motion were within 1%. Patient motion was most prevalent in the blood phase and MBF and MFR errors increased most substantially with motion in the inferior direction. Motion correction focused on these motions is needed to reduce MBF and MFR errors.

Novel Methods of Automated Quantification of Gap Junction Distribution and Interstitial Collagen Quantity from Animal and Human Atrial Tissue Sections

PubMed Central

Yan, Jiajie; Thomson, Justin K.; Wu, Xiaomin; Zhao, Weiwei; Pollard, Andrew E.; Ai, Xun

2014-01-01

Background Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies. Methods and Results Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and

Simplified quantification and whole-body distribution of [18F]FE-PE2I in nonhuman primates: prediction for human studies.

PubMed

Varrone, Andrea; Gulyás, Balázs; Takano, Akihiro; Stabin, Michael G; Jonsson, Cathrine; Halldin, Christer

2012-02-01

[(18)F]FE-PE2I is a promising dopamine transporter (DAT) radioligand. In nonhuman primates, we examined the accuracy of simplified quantification methods and the estimates of radiation dose of [(18)F]FE-PE2I. In the quantification study, binding potential (BP(ND)) values previously reported in three rhesus monkeys using kinetic and graphical analyses of [(18)F]FE-PE2I were used for comparison. BP(ND) using the cerebellum as reference region was obtained with four reference tissue methods applied to the [(18)F]FE-PE2I data that were compared with the kinetic and graphical analyses. In the whole-body study, estimates of adsorbed radiation were obtained in two cynomolgus monkeys. All reference tissue methods provided BP(ND) values within 5% of the values obtained with the kinetic and graphical analyses. The shortest imaging time for stable BP(ND) estimation was 54 min. The average effective dose of [(18)F]FE-PE2I was 0.021 mSv/MBq, similar to 2-deoxy-2-[(18)F]fluoro-d-glucose. The results in nonhuman primates suggest that [(18)F]FE-PE2I is suitable for accurate and stable DAT quantification, and its radiation dose estimates would allow for a maximal administered radioactivity of 476 MBq in human subjects. Copyright © 2012 Elsevier Inc. All rights reserved.

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

A liquid chromatography-tandem mass spectrometry-based targeted proteomics assay for monitoring P-glycoprotein levels in human breast tissue.

PubMed

Yang, Ting; Chen, Fei; Xu, Feifei; Wang, Fengliang; Xu, Qingqing; Chen, Yun

2014-09-25

P-glycoprotein (P-gp) can efflux drugs from cancer cells, and its overexpression is commonly associated with multi-drug resistance (MDR). Thus, the accurate quantification of P-gp would help predict the response to chemotherapy and for prognosis of breast cancer patients. An advanced liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based targeted proteomics assay was developed and validated for monitoring P-gp levels in breast tissue. Tryptic peptide 368IIDNKPSIDSYSK380 was selected as a surrogate analyte for quantification, and immuno-depleted tissue extract was used as a surrogate matrix. Matched pairs of breast tissue samples from 60 patients who were suspected to have drug resistance were subject to analysis. The levels of P-gp were quantified. Using data from normal tissue, we suggested a P-gp reference interval. The experimental values of tumor tissue samples were compared with those obtained from Western blotting and immunohistochemistry (IHC). The result indicated that the targeted proteomics approach was comparable to IHC but provided a lower limit of quantification (LOQ) and could afford more reliable results at low concentrations than the other two methods. LC/MS/MS-based targeted proteomics may allow the quantification of P-gp in breast tissue in a more accurate manner. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

Rauh, Juliane; Jacobi, Angela; Stiehler, Maik

2015-02-01

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

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

PubMed Central

Rauh, Juliane; Jacobi, Angela

2015-01-01

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

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. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2013-01-01

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

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

The characterization and certification of a quantitative reference material for Legionella detection and quantification by qPCR.

PubMed

Baume, M; Garrelly, L; Facon, J P; Bouton, S; Fraisse, P O; Yardin, C; Reyrolle, M; Jarraud, S

2013-06-01

The characterization and certification of a Legionella DNA quantitative reference material as a primary measurement standard for Legionella qPCR. Twelve laboratories participated in a collaborative certification campaign. A candidate reference DNA material was analysed through PCR-based limiting dilution assays (LDAs). The validated data were used to statistically assign both a reference value and an associated uncertainty to the reference material. This LDA method allowed for the direct quantification of the amount of Legionella DNA per tube in genomic units (GU) and the determination of the associated uncertainties. This method could be used for the certification of all types of microbiological standards for qPCR. The use of this primary standard will improve the accuracy of Legionella qPCR measurements and the overall consistency of these measurements among different laboratories. The extensive use of this certified reference material (CRM) has been integrated in the French standard NF T90-471 (April 2010) and in the ISO Technical Specification 12 869 (Anon 2012 International Standardisation Organisation) for validating qPCR methods and ensuring the reliability of these methods. © 2013 The Society for Applied Microbiology.

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

PubMed

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

2012-01-01

This report describes our efforts on quantification of tissue metabolite concentrations in mM by nuclear Overhauser enhanced and proton decoupled (13) C magnetic resonance spectroscopy and the Electric Reference To access In vivo Concentrations (ERETIC) method. Previous work showed that a calibrated synthetic magnetic resonance spectroscopy-like signal transmitted through an optical fiber and inductively coupled into a transmit/receive coil represents a reliable reference standard for in vivo (1) H magnetic resonance spectroscopy quantification on a clinical platform. In this work, we introduce a related implementation that enables simultaneous proton decoupling and ERETIC-based metabolite quantification and hence extends the applicability of the ERETIC method to nuclear Overhauser enhanced and proton decoupled in vivo (13) C magnetic resonance spectroscopy. In addition, ERETIC signal stability under the influence of simultaneous proton decoupling is investigated. The proposed quantification method was cross-validated against internal and external reference standards on human skeletal muscle. The ERETIC signal intensity stability was 100.65 ± 4.18% over 3 months including measurements with and without proton decoupling. Glycogen and unsaturated fatty acid concentrations measured with the ERETIC method were in excellent agreement with internal creatine and external phantom reference methods, showing a difference of 1.85 ± 1.21% for glycogen and 1.84 ± 1.00% for unsaturated fatty acid between ERETIC and creatine-based quantification, whereas the deviations between external reference and creatine-based quantification are 6.95 ± 9.52% and 3.19 ± 2.60%, respectively. Copyright © 2011 Wiley Periodicals, Inc.

Automated and Adaptable Quantification of Cellular Alignment from Microscopic Images for Tissue Engineering Applications

PubMed Central

Xu, Feng; Beyazoglu, Turker; Hefner, Evan; Gurkan, Umut Atakan

2011-01-01

Cellular alignment plays a critical role in functional, physical, and biological characteristics of many tissue types, such as muscle, tendon, nerve, and cornea. Current efforts toward regeneration of these tissues include replicating the cellular microenvironment by developing biomaterials that facilitate cellular alignment. To assess the functional effectiveness of the engineered microenvironments, one essential criterion is quantification of cellular alignment. Therefore, there is a need for rapid, accurate, and adaptable methodologies to quantify cellular alignment for tissue engineering applications. To address this need, we developed an automated method, binarization-based extraction of alignment score (BEAS), to determine cell orientation distribution in a wide variety of microscopic images. This method combines a sequenced application of median and band-pass filters, locally adaptive thresholding approaches and image processing techniques. Cellular alignment score is obtained by applying a robust scoring algorithm to the orientation distribution. We validated the BEAS method by comparing the results with the existing approaches reported in literature (i.e., manual, radial fast Fourier transform-radial sum, and gradient based approaches). Validation results indicated that the BEAS method resulted in statistically comparable alignment scores with the manual method (coefficient of determination R2=0.92). Therefore, the BEAS method introduced in this study could enable accurate, convenient, and adaptable evaluation of engineered tissue constructs and biomaterials in terms of cellular alignment and organization. PMID:21370940

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

Impact of time-of-flight PET on quantification errors in MR imaging-based attenuation correction.

PubMed

Mehranian, Abolfazl; Zaidi, Habib

2015-04-01

Time-of-flight (TOF) PET/MR imaging is an emerging imaging technology with great capabilities offered by TOF to improve image quality and lesion detectability. We assessed, for the first time, the impact of TOF image reconstruction on PET quantification errors induced by MR imaging-based attenuation correction (MRAC) using simulation and clinical PET/CT studies. Standard 4-class attenuation maps were derived by segmentation of CT images of 27 patients undergoing PET/CT examinations into background air, lung, soft-tissue, and fat tissue classes, followed by the assignment of predefined attenuation coefficients to each class. For each patient, 4 PET images were reconstructed: non-TOF and TOF both corrected for attenuation using reference CT-based attenuation correction and the resulting 4-class MRAC maps. The relative errors between non-TOF and TOF MRAC reconstructions were compared with their reference CT-based attenuation correction reconstructions. The bias was locally and globally evaluated using volumes of interest (VOIs) defined on lesions and normal tissues and CT-derived tissue classes containing all voxels in a given tissue, respectively. The impact of TOF on reducing the errors induced by metal-susceptibility and respiratory-phase mismatch artifacts was also evaluated using clinical and simulation studies. Our results show that TOF PET can remarkably reduce attenuation correction artifacts and quantification errors in the lungs and bone tissues. Using classwise analysis, it was found that the non-TOF MRAC method results in an error of -3.4% ± 11.5% in the lungs and -21.8% ± 2.9% in bones, whereas its TOF counterpart reduced the errors to -2.9% ± 7.1% and -15.3% ± 2.3%, respectively. The VOI-based analysis revealed that the non-TOF and TOF methods resulted in an average overestimation of 7.5% and 3.9% in or near lung lesions (n = 23) and underestimation of less than 5% for soft tissue and in or near bone lesions (n = 91). Simulation results showed that

Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-mass Spectrometry for Targeted and Microdomain-specific Protein Quantification in Human Postmortem Brain Tissue*

PubMed Central

MacDonald, Matthew L.; Ciccimaro, Eugene; Prakash, Amol; Banerjee, Anamika; Seeholzer, Steven H.; Blair, Ian A.; Hahn, Chang-Gyu

2012-01-01

Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease. PMID:22942359

PET Quantification of the Norepinephrine Transporter in Human Brain with (S,S)-18F-FMeNER-D2.

PubMed

Moriguchi, Sho; Kimura, Yasuyuki; Ichise, Masanori; Arakawa, Ryosuke; Takano, Harumasa; Seki, Chie; Ikoma, Yoko; Takahata, Keisuke; Nagashima, Tomohisa; Yamada, Makiko; Mimura, Masaru; Suhara, Tetsuya

2017-07-01

Norepinephrine transporter (NET) in the brain plays important roles in human cognition and the pathophysiology of psychiatric disorders. Two radioligands, ( S , S )- 11 C-MRB and ( S , S )- 18 F-FMeNER-D 2 , have been used for imaging NETs in the thalamus and midbrain (including locus coeruleus) using PET in humans. However, NET density in the equally important cerebral cortex has not been well quantified because of unfavorable kinetics with ( S , S )- 11 C-MRB and defluorination with ( S , S )- 18 F-FMeNER-D 2 , which can complicate NET quantification in the cerebral cortex adjacent to the skull containing defluorinated 18 F radioactivity. In this study, we have established analysis methods of quantification of NET density in the brain including the cerebral cortex using ( S , S )- 18 F-FMeNER-D 2 PET. Methods: We analyzed our previous ( S , S )- 18 F-FMeNER-D 2 PET data of 10 healthy volunteers dynamically acquired for 240 min with arterial blood sampling. The effects of defluorination on the NET quantification in the superficial cerebral cortex was evaluated by establishing a time stability of NET density estimations with an arterial input 2-tissue-compartment model, which guided the less-invasive reference tissue model and area under the time-activity curve methods to accurately quantify NET density in all brain regions including the cerebral cortex. Results: Defluorination of ( S , S )- 18 F-FMeNER-D 2 became prominent toward the latter half of the 240-min scan. Total distribution volumes in the superficial cerebral cortex increased with the scan duration beyond 120 min. We verified that 90-min dynamic scans provided a sufficient amount of data for quantification of NET density unaffected by defluorination. Reference tissue model binding potential values from the 90-min scan data and area under the time-activity curve ratios of 70- to 90-min data allowed for the accurate quantification of NET density in the cerebral cortex. Conclusion: We have established

Update on Controls for Isolation and Quantification Methodology of Extracellular Vesicles Derived from Adipose Tissue Mesenchymal Stem Cells

PubMed Central

Franquesa, Marcella; Hoogduijn, Martin J.; Ripoll, Elia; Luk, Franka; Salih, Mahdi; Betjes, Michiel G. H.; Torras, Juan; Baan, Carla C.; Grinyó, Josep M.; Merino, Ana Maria

2014-01-01

The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 μm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV. PMID:25374572

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.

Accurate determination of brain metabolite concentrations using ERETIC as external reference.

PubMed

Zoelch, Niklaus; Hock, Andreas; Heinzer-Schweizer, Susanne; Avdievitch, Nikolai; Henning, Anke

2017-08-01

Magnetic Resonance Spectroscopy (MRS) can provide in vivo metabolite concentrations in standard concentration units if a reliable reference signal is available. For 1 H MRS in the human brain, typically the signal from the tissue water is used as the (internal) reference signal. However, a concentration determination based on the tissue water signal most often requires a reliable estimate of the water concentration present in the investigated tissue. Especially in clinically interesting cases, this estimation might be difficult. To avoid assumptions about the water in the investigated tissue, the Electric REference To access In vivo Concentrations (ERETIC) method has been proposed. In this approach, the metabolite signal is compared with a reference signal acquired in a phantom and potential coil-loading differences are corrected using a synthetic reference signal. The aim of this study, conducted with a transceiver quadrature head coil, was to increase the accuracy of the ERETIC method by correcting the influence of spatial B 1 inhomogeneities and to simplify the quantification with ERETIC by incorporating an automatic phase correction for the ERETIC signal. Transmit field ( B1+) differences are minimized with a volume-selective power optimization, whereas reception sensitivity changes are corrected using contrast-minimized images of the brain and by adapting the voxel location in the phantom measurement closely to the position measured in vivo. By applying the proposed B 1 correction scheme, the mean metabolite concentrations determined with ERETIC in 21 healthy subjects at three different positions agree with concentrations derived with the tissue water signal as reference. In addition, brain water concentrations determined with ERETIC were in agreement with estimations derived using tissue segmentation and literature values for relative water densities. Based on the results, the ERETIC method presented here is a valid tool to derive in vivo metabolite

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

PubMed Central

2011-01-01

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

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

PubMed

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

2003-01-01

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

Three new mussel tissue standard reference materials (SRMs) for the determination of organic contaminants.

PubMed

Poster, Dianne L; Schantz, Michele M; Kucklick, John R; Lopez de Alda, Maria J; Porter, Barbara J; Pugh, Rebecca; Wise, Stephen A

2004-03-01

Three new mussel tissue standard reference materials (SRMs) have been developed by the National Institute of Standards and Technology (NIST) for the determination of the concentrations of organic contaminants. The most recently prepared material, SRM 1974b, is a fresh frozen tissue homogenate prepared from mussels ( Mytilus edulis) collected in Boston Harbor, Massachusetts. The other two materials, SRMs 2977 and 2978, are freeze-dried tissue homogenates prepared from mussels collected in Guanabara Bay, Brazil and Raritan Bay, New Jersey, respectively. All three new mussel tissue SRMs complement the current suite of marine natural-matrix SRMs available from NIST that are characterized for a wide range of contaminants (organic and inorganic). SRM 1974b has been developed to replace its predecessor SRM 1974a, Organics in Mussel Tissue, for which the supply is depleted. Similarly, SRMs 2977 and 2978 were developed to replace a previously available (supply depleted) freeze-dried version of SRM 1974a, SRM 2974, Organics in Freeze-Dried Mussel Tissue. SRM 1974b is the third in a series of fresh frozen mussel tissue homogenate SRMs prepared from mussels collected in Boston Harbor starting in 1988. SRM 1974b has certified concentration values for 22 polycyclic aromatic hydrocarbons (PAHs), 31 polychlorinated biphenyl congeners (PCBs), and 7 chlorinated pesticides. Reference values are provided for additional constituents: 16 PAHs, 8 PCBs plus total PCBs, 6 pesticides, total extractable organics, methylmercury, and 11 trace elements. PAH concentrations range from about 2 ng g(-1 )dry mass (cyclopenta[ cd]pyrene) to 180 ng g(-1 )dry mass (pyrene). PCB concentrations range from about 2 ng g(-1 )dry mass (PCB 157) to 120 ng g(-1 )dry mass (PCB 153). The reference value for total PCBs in SRM 1974b is (2020 +/- 420) ng g(-1 )dry mass. Pesticide concentrations range from about 4 ng g(-1 )dry mass (4,4'-DDT) to 40 ng g(-1 )dry mass (4,4'-DDE). SRM 2977 has certified values for 14

MicroRNA Expression in Formalin-fixed Paraffin-embedded Cancer Tissue: Identifying Reference MicroRNAs and Variability.

PubMed

Boisen, Mogens Karsbøl; Dehlendorff, Christian; Linnemann, Dorte; Schultz, Nicolai Aagaard; Jensen, Benny Vittrup; Høgdall, Estrid Vilma Solyom; Johansen, Julia Sidenius

2015-12-29

Archival formalin-fixed paraffin-embedded (FFPE) cancer tissue samples are a readily available resource for microRNA (miRNA) biomarker identification. No established standard for reference miRNAs in FFPE tissue exists. We sought to identify stable reference miRNAs for normalization of miRNA expression in FFPE tissue samples from patients with colorectal (CRC) and pancreatic (PC) cancer and to quantify the variability associated with sample age and fixation. High-throughput miRNA profiling results from 203 CRC and 256 PC FFPE samples as well as from 37 paired frozen/FFPE samples from nine other CRC tumors (methodological samples) were used. Candidate reference miRNAs were identified by their correlation with global mean expression. The stability of reference genes was analyzed according to published methods. The association between sample age and global mean miRNA expression was tested using linear regression. Variability was described using correlation coefficients and linear mixed effects models. Normalization effects were determined by changes in standard deviation and by hierarchical clustering. We created lists of 20 miRNAs with the best correlation to global mean expression in each cancer type. Nine of these miRNAs were present in both lists, and miR-103a-3p was the most stable reference miRNA for both CRC and PC FFPE tissue. The optimal number of reference miRNAs was 4 in CRC and 10 in PC. Sample age had a significant effect on global miRNA expression in PC (50% reduction over 20 years) but not in CRC. Formalin fixation for 2-6 days decreased miRNA expression 30-65%. Normalization using global mean expression reduced variability for technical and biological replicates while normalization using the expression of the identified reference miRNAs reduced variability only for biological replicates. Normalization only had a minor impact on clustering results. We identified suitable reference miRNAs for future miRNA expression experiments using CRC- and PC FFPE

Generic method for the absolute quantification of glutathione S-conjugates: Application to the conjugates of acetaminophen, clozapine and diclofenac.

PubMed

den Braver, Michiel W; Vermeulen, Nico P E; Commandeur, Jan N M

2017-03-01

Modification of cellular macromolecules by reactive drug metabolites is considered to play an important role in the initiation of tissue injury by many drugs. Detection and identification of reactive intermediates is often performed by analyzing the conjugates formed after trapping by glutathione (GSH). Although sensitivity of modern mass spectrometrical methods is extremely high, absolute quantification of GSH-conjugates is critically dependent on the availability of authentic references. Although 1 H NMR is currently the method of choice for quantification of metabolites formed biosynthetically, its intrinsically low sensitivity can be a limiting factor in quantification of GSH-conjugates which generally are formed at low levels. In the present study, a simple but sensitive and generic method for absolute quantification of GSH-conjugates is presented. The method is based on quantitative alkaline hydrolysis of GSH-conjugates and subsequent quantification of glutamic acid and glycine by HPLC after precolumn derivatization with o-phthaldialdehyde/N-acetylcysteine (OPA/NAC). Because of the lower stability of the glycine OPA/NAC-derivate, quantification of the glutamic acid OPA/NAC-derivate appeared most suitable for quantification of GSH-conjugates. The novel method was used to quantify the concentrations of GSH-conjugates of diclofenac, clozapine and acetaminophen and quantification was consistent with 1 H NMR, but with a more than 100-fold lower detection limit for absolute quantification. Copyright © 2017. Published by Elsevier B.V.

A candidate reference method using ICP-MS for sweat chloride quantification.

PubMed

Collie, Jake T; Massie, R John; Jones, Oliver A H; Morrison, Paul D; Greaves, Ronda F

2016-04-01

The aim of the study was to develop a method for sweat chloride (Cl) quantification using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to present to the Joint Committee for Traceability in Laboratory Medicine (JCTLM) as a candidate reference method for the diagnosis of cystic fibrosis (CF). Calibration standards were prepared from sodium chloride (NaCl) to cover the expected range of sweat Cl values. Germanium (Ge) and scandium (Sc) were selected as on-line (instrument based) internal standards (IS) and gallium (Ga) as the off-line (sample based) IS. The method was validated through linearity, accuracy and imprecision studies as well as enrolment into the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) for sweat electrolyte testing. Two variations of the ICP-MS method were developed, an on-line and off-line IS, and compared. Linearity was determined up to 225 mmol/L with a limit of quantitation of 7.4 mmol/L. The off-line IS demonstrated increased accuracy through the RCPAQAP performance assessment (CV of 1.9%, bias of 1.5 mmol/L) in comparison to the on-line IS (CV of 8.0%, bias of 3.8 mmol/L). Paired t-tests confirmed no significant differences between sample means of the two IS methods (p=0.53) or from each method against the RCPAQAP target values (p=0.08 and p=0.29). Both on and off-line IS methods generated highly reproducible results and excellent linear comparison to the RCPAQAP target results. ICP-MS is a highly accurate method with a low limit of quantitation for sweat Cl analysis and should be recognised as a candidate reference method for the monitoring and diagnosis of CF. Laboratories that currently practice sweat Cl analysis using ICP-MS should include an off-line IS to help negate any pre-analytical errors.

Choice of reference measurements affects quantification of long diffusion time behaviour using stimulated echoes.

PubMed

Kleinnijenhuis, Michiel; Mollink, Jeroen; Lam, Wilfred W; Kinchesh, Paul; Khrapitchev, Alexandre A; Smart, Sean C; Jbabdi, Saad; Miller, Karla L

2018-02-01

To demonstrate how reference data affect the quantification of the apparent diffusion coefficient (ADC) in long diffusion time measurements with diffusion-weighted stimulated echo acquisition mode (DW-STEAM) measurements, and to present a modification to avoid contribution from crusher gradients in DW-STEAM. For DW-STEAM, reference measurements at long diffusion times have significant b 0 value, because b = 0 cannot be achieved in practice as a result of the need for signal spoiling. Two strategies for acquiring reference data over a range of diffusion times were considered: constant diffusion weighting (fixed-b 0 ) and constant gradient area (fixed-q 0 ). Fixed-b 0 and fixed-q 0 were compared using signal calculations for systems with one and two diffusion coefficients, and experimentally using data from postmortem human corpus callosum samples. Calculations of biexponential diffusion decay show that the ADC is underestimated for reference images with b > 0, which can induce an apparent time-dependence for fixed-q 0 . Restricted systems were also found to be affected. Experimentally, the exaggeration of the diffusion time-dependent effect under fixed-q 0 versus fixed-b 0 was in a range predicted theoretically, accounting for 62% (longitudinal) and 35% (radial) of the time dependence observed in white matter. Variation in the b-value of reference measurements in DW-STEAM can induce artificial diffusion time dependence in ADC, even in the absence of restriction. Magn Reson Med 79:952-959, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of

Validation of reference genes for real-time quantitative PCR normalization in soybean developmental and germinating seeds.

PubMed

Li, Qing; Fan, Cheng-Ming; Zhang, Xiao-Mei; Fu, Yong-Fu

2012-10-01

Most of traditional reference genes chosen for real-time quantitative PCR normalization were assumed to be ubiquitously and constitutively expressed in vegetative tissues. However, seeds show distinct transcriptomes compared with the vegetative tissues. Therefore, there is a need for re-validation of reference genes in samples of seed development and germination, especially for soybean seeds. In this study, we aimed at identifying reference genes suitable for the quantification of gene expression level in soybean seeds. In order to identify the best reference genes for soybean seeds, 18 putative reference genes were tested with various methods in different seed samples. We combined the outputs of both geNorm and NormFinder to assess the expression stability of these genes. The reference genes identified as optimums for seed development were TUA5 and UKN2, whereas for seed germination they were novel reference genes Glyma05g37470 and Glyma08g28550. Furthermore, for total seed samples it was necessary to combine four genes of Glyma05g37470, Glyma08g28550, Glyma18g04130 and UKN2 [corrected] for normalization. Key message We identified several reference genes that stably expressed in soybean seed developmental and germinating processes.

Exome copy number variation detection: Use of a pool of unrelated healthy tissue as reference sample.

PubMed

Wenric, Stephane; Sticca, Tiberio; Caberg, Jean-Hubert; Josse, Claire; Fasquelle, Corinne; Herens, Christian; Jamar, Mauricette; Max, Stéphanie; Gothot, André; Caers, Jo; Bours, Vincent

2017-01-01

An increasing number of bioinformatic tools designed to detect CNVs (copy number variants) in tumor samples based on paired exome data where a matched healthy tissue constitutes the reference have been published in the recent years. The idea of using a pool of unrelated healthy DNA as reference has previously been formulated but not thoroughly validated. As of today, the gold standard for CNV calling is still aCGH but there is an increasing interest in detecting CNVs by exome sequencing. We propose to design a metric allowing the comparison of two CNV profiles, independently of the technique used and assessed the validity of using a pool of unrelated healthy DNA instead of a matched healthy tissue as reference in exome-based CNV detection. We compared the CNV profiles obtained with three different approaches (aCGH, exome sequencing with a matched healthy tissue as reference, exome sequencing with a pool of eight unrelated healthy tissue as reference) on three multiple myeloma samples. We show that the usual analyses performed to compare CNV profiles (deletion/amplification ratios and CNV size distribution) lack in precision when confronted with low LRR values, as they only consider the binary status of each CNV. We show that the metric-based distance constitutes a more accurate comparison of two CNV profiles. Based on these analyses, we conclude that a reliable picture of CNV alterations in multiple myeloma samples can be obtained from whole-exome sequencing in the absence of a matched healthy sample. © 2016 WILEY PERIODICALS, INC.

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

PubMed Central

Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses. PMID:26863232

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species.

PubMed

Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

Selection and validation of suitable reference genes for miRNA expression normalization by quantitative RT-PCR in citrus somatic embryogenic and adult tissues.

PubMed

Kou, Shu-Jun; Wu, Xiao-Meng; Liu, Zheng; Liu, Yuan-Long; Xu, Qiang; Guo, Wen-Wu

2012-12-01

miRNAs have recently been reported to modulate somatic embryogenesis (SE), a key pathway of plant regeneration in vitro. For expression level detection and subsequent function dissection of miRNAs in certain biological processes, qRT-PCR is one of the most effective and sensitive techniques, for which suitable reference gene selection is a prerequisite. In this study, three miRNAs and eight non-coding RNAs (ncRNA) were selected as reference candidates, and their expression stability was inspected in developing citrus SE tissues cultured at 20, 25, and 30 °C. Stability of the eight non-miRNA ncRNAs was further validated in five adult tissues without temperature treatment. The best single reference gene for SE tissues was snoR14 or snoRD25, while for the adult tissues the best one was U4; although they were not as stable as the optimal multiple references snoR14 + U6 for SE tissues and snoR14 + U5 for adult tissues. For expression normalization of less abundant miRNAs in SE tissues, miR3954 was assessed as a viable reference. Single reference gene snoR14 outperformed multiple references for the overall SE and adult tissues. As one of the pioneer systematic studies on reference gene identification for plant miRNA normalization, this study benefits future exploration on miRNA function in citrus and provides valuable information for similar studies in other higher plants. Three miRNAs and eight non-coding RNAs were tested as reference candidates on developing citrus SE tissues. Best single references snoR14 or snoRD25 and optimal multiple references snoR14 + U6, snoR14 + U5 were identified.

Hyperplex-MRM: a hybrid multiple reaction monitoring method using mTRAQ/iTRAQ labeling for multiplex absolute quantification of human colorectal cancer biomarker.

PubMed

Yin, Hong-Rui; Zhang, Lei; Xie, Li-Qi; Huang, Li-Yong; Xu, Ye; Cai, San-Jun; Yang, Peng-Yuan; Lu, Hao-Jie

2013-09-06

Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.

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. © 2016 Wiley Periodicals, Inc.

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.

Evaluation of Reference Genes for Quantitative Real-Time PCR in Songbirds

PubMed Central

Zinzow-Kramer, Wendy M.; Horton, Brent M.; Maney, Donna L.

2014-01-01

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbird: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology. PMID:24780145

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

PubMed Central

Ohno, Misa; Togashi, Yuto; Tsuda, Kyoko; Okawa, Kazuaki; Kamaya, Minori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

2013-01-01

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific. PMID:23826286

Quantification of γ-aminobutyric acid (GABA) in 1 H MRS volumes composed heterogeneously of grey and white matter.

PubMed

Mikkelsen, Mark; Singh, Krish D; Brealy, Jennifer A; Linden, David E J; Evans, C John

2016-11-01

The quantification of γ-aminobutyric acid (GABA) concentration using localised MRS suffers from partial volume effects related to differences in the intrinsic concentration of GABA in grey (GM) and white (WM) matter. These differences can be represented as a ratio between intrinsic GABA in GM and WM: r M . Individual differences in GM tissue volume can therefore potentially drive apparent concentration differences. Here, a quantification method that corrects for these effects is formulated and empirically validated. Quantification using tissue water as an internal concentration reference has been described previously. Partial volume effects attributed to r M can be accounted for by incorporating into this established method an additional multiplicative correction factor based on measured or literature values of r M weighted by the proportion of GM and WM within tissue-segmented MRS volumes. Simulations were performed to test the sensitivity of this correction using different assumptions of r M taken from previous studies. The tissue correction method was then validated by applying it to an independent dataset of in vivo GABA measurements using an empirically measured value of r M . It was shown that incorrect assumptions of r M can lead to overcorrection and inflation of GABA concentration measurements quantified in volumes composed predominantly of WM. For the independent dataset, GABA concentration was linearly related to GM tissue volume when only the water signal was corrected for partial volume effects. Performing a full correction that additionally accounts for partial volume effects ascribed to r M successfully removed this dependence. With an appropriate assumption of the ratio of intrinsic GABA concentration in GM and WM, GABA measurements can be corrected for partial volume effects, potentially leading to a reduction in between-participant variance, increased power in statistical tests and better discriminability of true effects. Copyright © 2016 John

Virtual touch tissue quantification using acoustic radiation force impulse technology: initial clinical experience with solid breast masses.

PubMed

Bai, Min; Du, Lianfang; Gu, Jiying; Li, Fan; Jia, Xiao

2012-02-01

The purpose of this study was to investigate the clinical usage of Virtual Touch tissue quantification (VTQ; Siemens Medical Solutions, Mountain View, CA) implementing sonographic acoustic radiation force impulse technology for differentiation between benign and malignant solid breast masses. A total of 143 solid breast masses were examined with VTQ, and their shear wave velocities (SWVs) were measured. From all of the masses, 30 were examined by two independent operators to evaluate the reproducibility of the results of VTQ measurement. All masses were later surgically resected, and the histologic results were correlated with the SWV results. A receiver operating characteristic curve was calculated to assess the diagnostic performance of VTQ. A total of 102 benign lesions and 41 carcinomas were diagnosed on the basis of histologic examination. The VTQ measurements performed by the two independent operators yielded a correlation coefficient of 0.885. Applying a cutoff point of 3.065 m/s, a significant difference (P < .001) was found between the SWVs of the benign (mean ± SD, 2.25 ± 0.59 m/s) and malignant (5.96 ± 2.96 m/s) masses. The sensitivity, specificity, and area under the receiver operating characteristic curve for the differentiation were 75.6%, 95.1%, and 85.6%, respectively. When the repeated non-numeric result X.XX of the SWV measurements was designated as an indicator of malignancy, the sensitivity, specificity, and accuracy were 63.4%, 100%, and 89.5%. Virtual Touch tissue quantification can yield reproducible and quantitative diagnostic information on solid breast masses and serve as an effective diagnostic tool for differentiation between benign and malignant solid masses.

Quantification of Toxoplasma gondii in tissue samples of experimentally infected goats by magnetic capture and real-time PCR.

PubMed

Juránková, Jana; Opsteegh, Marieke; Neumayerová, Helena; Kovařčík, Kamil; Frencová, Anita; Baláž, Vojtěch; Volf, Jiří; Koudela, Břetislav

2013-03-31

Undercooked meat containing tissue cysts is one of the most common sources of Toxoplasma gondii infection in humans. Goats are very susceptible to clinical toxoplasmosis, and especially kids are common food animals, thereby representing a risk for human infection. A sequence-specific magnetic capture method was used for isolation of T. gondii DNA from tissue samples from experimentally infected goat-kids and real-time PCR for the 529 bp repeat element allowed quantification of T. gondii DNA. The contamination level in different types of tissue and in two groups of goats euthanized 30 and 90 dpi was compared. The highest concentration of T. gondii DNA in both groups of goats was found in lung tissue, but only the higher parasite count in lung tissue compared to other organs in group A (euthanized 30 dpi) was statistically significant. T. gondii concentrations were higher in liver and dorsal muscle samples from goats euthanized 90 dpi than in goats euthanized at 30 dpi, while the T. gondii concentration in hearts decreased. This study describes for the first time distribution of T. gondii parasites in post-weaned goat kids. New information about T. gondii predilection sites in goats and about the progression of infection between 30 and 90 dpi was achieved. Copyright © 2012 Elsevier B.V. All rights reserved.

Role of Virtual Touch Tissue Quantification in Hashimoto's Thyroiditis.

PubMed

Lin, Zi-Mei; Wang, Yao; Liu, Chun-Mei; Yan, Cao-Xin; Huang, Pin-Tong

2018-06-01

We investigated the role of the virtual touch tissue quantification (VTQ) technique in diagnosing Hashimoto's thyroiditis (HT) and in distinguishing various HT-related thyroid dysfunctions. Two hundred HT patients and 100 healthy volunteers (the control group) were enrolled. The diagnostic performance of VTQ in predicting HT was calculated as the area under the receiver operating characteristic curve (A Z ). The HT patients were further classified into three subgroups on the basis of serologic tests of thyroid function: hyperthyroidism, euthyroidism and hypothyroidism. Comparisons of shear wave velocity (SWV) between three subgroups were evaluated by analysis of variance. The mean SWV of the control group was significantly lower than that of the HT group (1.93 ± 0.33 m/s vs. 2.32 ± 0.49 m/s, p <0.001). A z was 0.734 with a cut-off value of 1.86 m/s for performance of SWV in distinguishing between HT and a healthy thyroid; the sensitivity and specificity were 82.5% and 50.0%, respectively. Mean SWV values in the three HT subgroups (hyperthyroidism [2.07 ± 0.37 cm/s] vs. euthyroidism [2.20 ± 0.40 cm/s] vs. hypothyroidism [2.49 ± 0.46 cm/s]) were significantly different (p <0.05). Our results suggest that VTQ is a promising technique for assessing HT and HT-related thyroid dysfunction. Copyright © 2018 World Federation for Ultrasound in Medicine and Biology. Published by Elsevier Inc. All rights reserved.

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

Validation of a DIXON-based fat quantification technique for the measurement of visceral fat using a CT-based reference standard.

PubMed

Heckman, Katherine M; Otemuyiwa, Bamidele; Chenevert, Thomas L; Malyarenko, Dariya; Derstine, Brian A; Wang, Stewart C; Davenport, Matthew S

2018-06-27

The purpose of the study is to determine whether a novel semi-automated DIXON-based fat quantification algorithm can reliably quantify visceral fat using a CT-based reference standard. This was an IRB-approved retrospective cohort study of 27 subjects who underwent abdominopelvic CT within 7 days of proton density fat fraction (PDFF) mapping on a 1.5T MRI. Cross-sectional visceral fat area per slice (cm 2 ) was measured in blinded fashion in each modality at intervertebral disc levels from T12 to L4. CT estimates were obtained using a previously published semi-automated computational image processing system that sums pixels with attenuation - 205 to - 51 HU. MR estimates were obtained using two novel semi-automated DIXON-based fat quantification algorithms that measure visceral fat area by spatially regularizing non-uniform fat-only signal intensity or de-speckling PDFF 2D images and summing pixels with PDFF ≥ 50%. Pearson's correlations and Bland-Altman analyses were performed. Visceral fat area per slice ranged from 9.2 to 429.8 cm 2 for MR and from 1.6 to 405.5 cm 2 for CT. There was a strong correlation between CT and MR methods in measured visceral fat area across all studied vertebral body levels (r = 0.97; n = 101 observations); the least (r = 0.93) correlation was at T12. Bland-Altman analysis revealed a bias of 31.7 cm 2 (95% CI [- 27.1]-90.4 cm 2 ), indicating modestly higher visceral fat assessed by MR. MR- and CT-based visceral fat quantification are highly correlated and have good cross-modality reliability, indicating that visceral fat quantification by either method can yield a stable and reliable biomarker.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of Adipose Tissue Leukocytosis in Obesity

PubMed Central

Grant, Ryan; Youm, Yun-Hee; Ravussin, Anthony; Dixit, Vishwa Deep

2014-01-01

Summary The infiltration of immune cell subsets in adipose tissue termed ‘adipose tissue leukocytosis’ is a critical event in the development of chronic inflammation and obesity-associated comorbidities. Given that a significant proportion of cells in adipose tissue of obese patients are of hematopoietic lineage, the distinct adipose depots represent an uncharacterized immunological organ that can impact metabolic functions. Here, we describe approaches to characterize and isolate leukocytes from the complex adipose tissue microenvironment to aid mechanistic studies to understand the role of specific pattern recognition receptors (PRRs) such as inflammasomes in adipose-immune crosstalk. PMID:23852606

Establishing references for gene expression analyses by RT-qPCR in Theobroma cacao tissues.

PubMed

Pinheiro, T T; Litholdo, C G; Sereno, M L; Leal, G A; Albuquerque, P S B; Figueira, A

2011-11-17

Lack of continuous progress in Theobroma cacao (Malvaceae) breeding, especially associated with seed quality traits, requires more efficient selection methods based on genomic information. Reverse transcript quantitative PCR (RT-qPCR) has become the method of choice for gene expression analysis, but relative expression analysis requires various reference genes, which must be stable across various biological conditions. We sought suitable reference genes for various tissues of cacao, especially developing seeds. Ten potential reference genes were analyzed for stability at various stages of embryo development, leaves, stems, roots, flowers, and pod epicarp; seven of them were also evaluated in shoot tips treated either with hormones (salicylate; ethefon; methyl-jasmonate) or after inoculation with the fungus Moniliophthora perniciosa (Marasmiaceae sensu lato). For developing embryos, the three most stable genes were actin (ACT), polyubiquitin (PUB), and ribosomal protein L35 (Rpl35). In the analyses of various tissues, the most stable genes were malate dehydrogenase (MDH), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and acyl-carrier protein B (ACP B). GAPDH, MDH and tubulin (TUB) were the most appropriate for normalization when shoot apexes were treated with hormones, while ACT, TUB and Rpl35 were the most appropriate after inoculation with M. perniciosa. We conclude that for each plant system and biological or ontogenetical condition, there is a need to define suitable reference genes. This is the first report to define reference genes for expression studies in cacao.

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events.

PubMed

Demeke, Tigst; Eng, Monika

2018-05-01

Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences ( HMG-I/Y , FatA(A), CruA and Ccf) for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A), reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A) reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences) were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes.

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. Copyright © 2015 John Wiley & Sons, Ltd.

FDG-PET reproducibility in tumor-bearing mice: comparing a traditional SUV approach with a tumor-to-brain tissue ratio approach.

PubMed

Busk, Morten; Munk, Ole L; Jakobsen, Steen; Frøkiær, Jørgen; Overgaard, Jens; Horsman, Michael R

2017-05-01

Current [F-18]-fluorodeoxyglucose positron emission tomography (FDG-PET) procedures in tumor-bearing mice typically includes fasting, anesthesia, and standardized uptake value (SUV)-based quantification. Such procedures may be inappropriate for prolonged multiscan experiments. We hypothesize that normalization of tumor FDG retention relative to a suitable reference tissue may improve accuracy as this method may be less susceptible to uncontrollable day-to-day changes in blood glucose levels, physical activity, or unnoticed imperfect tail vein injections. Fed non-anesthetized tumor-bearing mice were administered FDG intravenously (i.v.) or intraperitoneally (i.p.) and PET scanned on consecutive days using a Mediso nanoScan PET/magnetic resonance imaging (MRI). Reproducibility of various PET-deduced measures of tumor FDG retention, including normalization to FDG signal in reference organs and a conventional SUV approach, was evaluated. Day-to-day variability in i.v. injected mice was lower when tumor FDG retention was normalized to brain signal (T/B), compared to normalization to other tissues or when using SUV-based normalization. Assessment of tissue radioactivity in dissected tissues confirmed the validity of PET-derived T/B ratios. Mean T/B and SUV values were similar in i.v. and i.p. administered animals, but SUV normalization was more robust in the i.p. group than in the i.v. group. Multimodality scanners allow tissue delineation and normalization of tumor FDG uptake relative to reference tissues. Normalization to brain, but not liver or kidney, improved scan reproducibility considerably and was superior to traditional SUV quantification in i.v. tracer-injected animals. Day-to-day variability in SUV's was lower in i.p. than in i.v. injected animals, and i.p. injections may therefore be a valuable alternative in prolonged rodent studies, where repeated vein injections are undesirable.

Quantification of right ventricular volume in dogs: a comparative study between three-dimensional echocardiography and computed tomography with the reference method magnetic resonance imaging.

PubMed

Sieslack, Anne K; Dziallas, Peter; Nolte, Ingo; Wefstaedt, Patrick; Hungerbühler, Stephan O

2014-10-12

Right ventricular (RV) volume and function are important diagnostic and prognostic factors in dogs with primary or secondary right-sided heart failure. The complex shape of the right ventricle and its retrosternal position make the quantification of its volume difficult. For that reason, only few studies exist, which deal with the determination of RV volume parameters. In human medicine cardiac magnetic resonance imaging (CMRI) is considered to be the reference technique for RV volumetric measurement (Nat Rev Cardiol 7(10):551-563, 2010), but cardiac computed tomography (CCT) and three-dimensional echocardiography (3DE) are other non-invasive methods feasible for RV volume quantification. The purpose of this study was the comparison of 3DE and CCT with CMRI, the gold standard for RV volumetric quantification. 3DE showed significant lower and CCT significant higher right ventricular volumes than CMRI. Both techniques showed very good correlations (R > 0.8) with CMRI for the volumetric parameters end-diastolic volume (EDV) and end-systolic volume (ESV). Ejection fraction (EF) and stroke volume (SV) were not different when considering CCT and CMRI, whereas 3DE showed a significant higher EF and lower SV than CMRI. The 3DE values showed excellent intra-observer variability (<3%) and still acceptable inter-observer variability (<13%). CCT provides an accurate image quality of the right ventricle with comparable results to the reference method CMRI. CCT overestimates the RV volumes; therefore, it is not an interchangeable method, having the disadvantage as well of needing general anaesthesia. 3DE underestimated the RV-Volumes, which could be explained by the worse image resolution. The excellent correlation between the methods indicates a close relationship between 3DE and CMRI although not directly comparable. 3DE is a promising technique for RV volumetric quantification, but further studies in awake dogs and dogs with heart disease are necessary to evaluate its

Towards absolute quantification of allergenic proteins in food--lysozyme in wine as a model system for metrologically traceable mass spectrometric methods and certified reference materials.

PubMed

Cryar, Adam; Pritchard, Caroline; Burkitt, William; Walker, Michael; O'Connor, Gavin; Burns, Duncan Thorburn; Quaglia, Milena

2013-01-01

Current routine food allergen quantification methods, which are based on immunochemistry, offer high sensitivity but can suffer from issues of specificity and significant variability of results. MS approaches have been developed, but currently lack metrological traceability. A feasibility study on the application of metrologically traceable MS-based reference procedures was undertaken. A proof of concept involving proteolytic digestion and isotope dilution MS for quantification of protein allergens in a food matrix was undertaken using lysozyme in wine as a model system. A concentration of lysozyme in wine of 0.95 +/- 0.03 microg/g was calculated based on the concentrations of two peptides, confirming that this type of analysis is viable at allergenically meaningful concentrations. The challenges associated with this promising method were explored; these included peptide stability, chemical modification, enzymatic digestion, and sample cleanup. The method is suitable for the production of allergen in food certified reference materials, which together with the achieved understanding of the effects of sample preparation and of the matrix on the final results, will assist in addressing the bias of the techniques routinely used and improve measurement confidence. Confirmation of the feasibility of MS methods for absolute quantification of an allergenic protein in a food matrix with results traceable to the International System of Units is a step towards meaningful comparison of results for allergen proteins among laboratories. This approach will also underpin risk assessment and risk management of allergens in the food industry, and regulatory compliance of the use of thresholds or action levels when adopted.

Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity

PubMed Central

Pérez-Pérez, Rafael; López, Juan A.; García-Santos, Eva; Camafeita, Emilio; Gómez-Serrano, María; Ortega-Delgado, Francisco J.; Ricart, Wifredo; Fernández-Real, José M.; Peral, Belén

2012-01-01

Background Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. Methodology and Findings To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. Conclusions ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity

Protein quantification using a cleavable reporter peptide.

PubMed

Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

2015-02-06

Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.

Reference tissue normalization in longitudinal (18)F-florbetapir positron emission tomography of late mild cognitive impairment.

PubMed

Shokouhi, Sepideh; Mckay, John W; Baker, Suzanne L; Kang, Hakmook; Brill, Aaron B; Gwirtsman, Harry E; Riddle, William R; Claassen, Daniel O; Rogers, Baxter P

2016-01-15

Semiquantitative methods such as the standardized uptake value ratio (SUVR) require normalization of the radiotracer activity to a reference tissue to monitor changes in the accumulation of amyloid-β (Aβ) plaques measured with positron emission tomography (PET). The objective of this study was to evaluate the effect of reference tissue normalization in a test-retest (18)F-florbetapir SUVR study using cerebellar gray matter, white matter (two different segmentation masks), brainstem, and corpus callosum as reference regions. We calculated the correlation between (18)F-florbetapir PET and concurrent cerebrospinal fluid (CSF) Aβ1-42 levels in a late mild cognitive impairment cohort with longitudinal PET and CSF data over the course of 2 years. In addition to conventional SUVR analysis using mean and median values of normalized brain radiotracer activity, we investigated a new image analysis technique-the weighted two-point correlation function (wS2)-to capture potentially more subtle changes in Aβ-PET data. Compared with the SUVRs normalized to cerebellar gray matter, all cerebral-to-white matter normalization schemes resulted in a higher inverse correlation between PET and CSF Aβ1-42, while the brainstem normalization gave the best results (high and most stable correlation). Compared with the SUVR mean and median values, the wS2 values were associated with the lowest coefficient of variation and highest inverse correlation to CSF Aβ1-42 levels across all time points and reference regions, including the cerebellar gray matter. The selection of reference tissue for normalization and the choice of image analysis method can affect changes in cortical (18)F-florbetapir uptake in longitudinal studies.

Reference gene stability of a synanthropic fly, Chrysomya megacephala.

PubMed

Wang, Xiaoyun; Xiong, Mei; Wang, Jialu; Lei, Chaoliang; Zhu, Fen

2015-10-29

Stable reference genes are essential for accurate normalization in gene expression studies with reverse transcription quantitative polymerase chain reaction (qPCR). A synanthropic fly, Chrysomya megacephala, is a well known medical vector and forensic indicator. Unfortunately, previous studies did not look at the stability of reference genes used in C. megacephala. In this study, the expression level of Actin, ribosomal protein L8 (Rpl8), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1), α-tubulin (α-TUB), β-tubulin (β-TUB), TATA binding box (TBP), 18S rRNA (18S) and ribosomal protein S7 (Rps7) were evaluated for their stability using online software RefFinder, which combines the normal software of the ΔCt method, BestKeeper, Normfinder, and geNorm. Moreover the number of suitable reference gene pairs was also suggested by Excel-based geNorm. The expression levels of these reference genes were evaluated under different experimental conditions with special perspectives of forensic applications: developmental stages (eggs, first, second and third instar larvae, pupae and adults); food sources of larvae (pork, fish and chicken); feeding larvae with drugs (untreated control, Estazolam and Marvelon); feeding larvae with heavy metals (untreated control, cadmium and zinc); tissues of adults (head, thorax, abdomen, legs and wings). According to RefFinder, EF1 was the most suitable reference gene of developmental stages, food and tissues; 18S and GAPDH were the most suitable reference genes for drugs and heavy metals, respectively, which could be widely used for quantification of target gene expression with qPCR in C. megacephala. Suitable reference gene pairs were also suggested by geNorm. This fundamental but vital work should facilitate the gene studies of related biological processes and deepen the understanding in physiology, toxicology, and especially medical and forensic entomology of C. megacephala.

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

PubMed

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

2013-08-01

In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of seven bioactive components including paeoniflorin, nobiletin, tangeretin, liquiritigenin, isoliquiritigenin, liquiritin and formononetin in rat plasma and tissues after oral administration of Si-Ni-San extract using astragaloside IV as internal standard (IS). The plasma and tissue samples were extracted by solid-phase extraction. Chromatographic separation was accomplished on a C18 column with a multiple-step gradient elution. The quantification was obtained by scanning with multiple reaction monitoring via an electrospray ionization source that was operated by switching between the positive and negative modes in two MS/MS scan segments. Full validation of the assay was implemented. In conclusion, this method demonstrated good linearity and specificity. The lower limits of quantification for the analytes were <7.5 ng/mL. Intra- and inter-day precisions (RSD) were <12.5% and accuracy (RE) ranged from -10.2 to 7.3%. The average recoveries of the analytes from rat plasma and tissues were >65.2% and 58.6%, respectively. The validated method was further applied to the determination of actual rat plasma and tissues after oral administration of Si-Ni-San extract. The results provided a meaningful basis for the clinical application of this prescription. Copyright © 2013 John Wiley & Sons, Ltd.

Characterization of epitope specificities of reference antibodies used for the quantification of the birch pollen allergen Bet v 1.

PubMed

Brier, S; Le Mignon, M; Jain, K; Lebrun, C; Peurois, F; Kellenberger, C; Bordas-Le Floch, V; Mascarell, L; Nony, E; Moingeon, P

2018-05-01

Accurate allergen quantification is needed to document the consistency of allergen extracts used for immunotherapy. Herein, we characterize the epitope specificities of two monoclonal antibodies used in an ELISA for the quantification of the major birch pollen allergen Bet v 1, established as a reference by the BSP090 European project. The ability of mAbs 5B4 and 6H4 to recognize Bet v 1 isoforms was addressed by immunochromatography. The capacity of each mAb to compete with patients' IgE for binding to Bet v 1 was measured by ELISA inhibition. Epitope mapping was performed by pepscan analysis, site-directed mutagenesis, and hydrogen/deuterium exchange-mass spectrometry. The 5B4 epitope corresponds to a peptide sequence (I56-K68) overlapping with the binding sites of patients' serum IgEs. Mutation of residues P59, E60, and K65 abolishes 5B4 binding to Bet v 1 and reduces the level of IgE recognition. In contrast, 6H4 recognizes a conformational epitope lying opposite to the 5B4 binding site, involving residues located in segments I44-K55 and R70-F79. Substitution of E45 reduces the binding capacity of 6H4, confirming that it is critical for the interaction. Both mAbs interact with >90% of Bet v 1 content present in the birch pollen extract, while displaying a weak cross-reactivity with other allergens of the PR-10 family. MAbs 5B4 and 6H4 recognize structurally distinct epitopes present in the vast majority of Bet v 1 isoforms. These results support the relevance as a reference method of the Bet v 1-specific quantitative ELISA adopted by the European Pharmacopoeia. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis.

PubMed

Nguyen, Duc Quan; Eamens, Andrew L; Grof, Christopher P L

2018-01-01

Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail ( Setaria viridis ) has recently been proposed as a potential experimental model for the study of C 4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens -mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis ( S. viridis ). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data. Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE / THERONINE - PROTEIN PHOSPHATASE 2A ( PP2A ), 5 '- ADENYLYLSULFATE REDUCTASE 6 ( ASPR6 ) and DUAL SPECIFICITY PHOSPHATASE ( DUSP ) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A , ASPR6 and DUSP , were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE ( CAD ) genes across the same tissues. This approach readily demonstrated the suitably of the three

Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

PubMed Central

2014-01-01

Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level

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).

A novel method to quantify IRDye800CW fluorescent antibody probes ex vivo in tissue distribution studies.

PubMed

Oliveira, Sabrina; Cohen, Ruth; Walsum, Marijke Stigter-van; van Dongen, Guus Ams; Elias, Sjoerd G; van Diest, Paul J; Mali, Willem; van Bergen En Henegouwen, Paul Mp

2012-09-25

We describe a new method for biodistribution studies with IRDye800CW fluorescent antibody probes. This method allows the quantification of the IRDye800CW fluorescent tracer in percentage of injected dose per gram of tissue (% ID/g), and it is herein compared to the generally used reference method that makes use of radioactivity. Cetuximab was conjugated to both the near-infrared fluorophore IRDye800CW and/or the positron emitter 89-zirconium, which was injected in nude mice bearing A431 human tumor xenografts. Positron emission tomography (PET) and optical imaging were performed 24 h post-injection (p.i.). For the biodistribution study, organs and tumors were collected 24 h p.i., and each of these was halved. One half was used for the determination of probe uptake by radioactivity measurement. The other half was homogenized, and the content of the fluorescent probe was determined by extrapolation from a calibration curve made with the injected probe. Tumors were clearly visualized with both modalities, and the calculated tumor-to-normal tissue ratios were very similar for optical and PET imaging: 3.31 ± 1.09 and 3.15 ± 0.99, respectively. Although some variations were observed in ex vivo analyses, tumor uptake was within the same range for IRDye800CW and gamma ray quantification: 15.07 ± 3.66% ID/g and 13.92 ± 2.59% ID/g, respectively. The novel method for quantification of the optical tracer IRDye800CW gives similar results as the reference method of gamma ray quantification. This new method is considered very useful in the context of the preclinical development of IRDye800CW fluorescent probes for optical molecular imaging, likely contributing to the selection of lead compounds that are the most promising for clinical translation.

Mammalian monoamine-oxidizing enzymes, with special reference to benzylamine oxidase in human tissues.

PubMed

Lewinsohn, R

1984-01-01

A review is presented of the monoamine-oxidizing enzymes with special reference to the activity of benzylamine oxidase (BzAO) in human tissues. Methods of study of amine oxidases, properties (chiefly of BzAO) and some problems concerning substrate and inhibitor specificity and multiple forms of monoamine oxidase (MAO) are surveyed. The substrate specificity of human plasma BzAO is compared with that of amine-oxidizing enzymes in plasma or serum of other species. Correlations of plasma BzAO and platelet MAO activity with clinical findings are discussed. The distribution of amine oxidase activities in solid human tissues is reviewed, in particular BzAO in blood vessels and richly-vascularized tissues, as well as kinetic constants and altered patterns of activity of BzAO in human atherosclerosis. Activities of the amine oxidases in non-vascular smooth muscle, in cultured cells, and in various tissues related to human gestation, are discussed. The present knowledge of BzAO is discussed in terms of its possible clinical relevance to several human disease states, and the importance of the enzyme in the human body.

Non-invasive quantification of tumour heterogeneity in water diffusivity to differentiate malignant from benign tissues of urinary bladder: a phase I study.

PubMed

Nguyen, Huyen T; Shah, Zarine K; Mortazavi, Amir; Pohar, Kamal S; Wei, Lai; Jia, Guang; Zynger, Debra L; Knopp, Michael V

2017-05-01

To quantify the heterogeneity of the tumour apparent diffusion coefficient (ADC) using voxel-based analysis to differentiate malignancy from benign wall thickening of the urinary bladder. Nineteen patients with histopathological findings of their cystectomy specimen were included. A data set of voxel-based ADC values was acquired for each patient's lesion. Histogram analysis was performed on each data set to calculate uniformity (U) and entropy (E). The k-means clustering of the voxel-wised ADC data set was implemented to measure mean intra-cluster distance (MICD) and largest inter-cluster distance (LICD). Subsequently, U, E, MICD, and LICD for malignant tumours were compared with those for benign lesions using a two-sample t-test. Eleven patients had pathological confirmation of malignancy and eight with benign wall thickening. Histogram analysis showed that malignant tumours had a significantly higher degree of ADC heterogeneity with lower U (P = 0.016) and higher E (P = 0.005) than benign lesions. In agreement with these findings, k-means clustering of voxel-wise ADC indicated that bladder malignancy presented with significantly higher MICD (P < 0.001) and higher LICD (P = 0.002) than benign wall thickening. The quantitative assessment of tumour diffusion heterogeneity using voxel-based ADC analysis has the potential to become a non-invasive tool to distinguish malignant from benign tissues of urinary bladder cancer. • Heterogeneity is an intrinsic characteristic of tumoral tissue. • Non-invasive quantification of tumour heterogeneity can provide adjunctive information to improve cancer diagnosis accuracy. • Histogram analysis and k-means clustering can quantify tumour diffusion heterogeneity. • The quantification helps differentiate malignant from benign urinary bladder tissue.

Reference-free determination of tissue absorption coefficient by modulation transfer function characterization in spatial frequency domain.

PubMed

Chen, Weiting; Zhao, Huijuan; Li, Tongxin; Yan, Panpan; Zhao, Kuanxin; Qi, Caixia; Gao, Feng

2017-08-08

Spatial frequency domain (SFD) measurement allows rapid and non-contact wide-field imaging of the tissue optical properties, thus has become a potential tool for assessing physiological parameters and therapeutic responses during photodynamic therapy of skin diseases. The conventional SFD measurement requires a reference measurement within the same experimental scenario as that for a test one to calibrate mismatch between the real measurements and the model predictions. Due to the individual physical and geometrical differences among different tissues, organs and patients, an ideal reference measurement might be unavailable in clinical trials. To address this problem, we present a reference-free SFD determination of absorption coefficient that is based on the modulation transfer function (MTF) characterization. Instead of the absolute amplitude that is used in the conventional SFD approaches, we herein employ the MTF to characterize the propagation of the modulated lights in tissues. With such a dimensionless relative quantity, the measurements can be naturally corresponded to the model predictions without calibrating the illumination intensity. By constructing a three-dimensional database that portrays the MTF as a function of the optical properties (both the absorption coefficient μ a and the reduced scattering coefficient [Formula: see text]) and the spatial frequency, a look-up table approach or a least-square curve-fitting method is readily applied to recover the absorption coefficient from a single frequency or multiple frequencies, respectively. Simulation studies have verified the feasibility of the proposed reference-free method and evaluated its accuracy in the absorption recovery. Experimental validations have been performed on homogeneous tissue-mimicking phantoms with μ a ranging from 0.01 to 0.07 mm -1 and [Formula: see text] = 1.0 or 2.0 mm -1 . The results have shown maximum errors of 4.86 and 7% for [Formula: see text] = 1.0 mm -1 and

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

PubMed

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

2007-05-01

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

Quantification of extracellular matrix expansion by CMR in infiltrative heart disease.

PubMed

Mongeon, François-Pierre; Jerosch-Herold, Michael; Coelho-Filho, Otávio Rizzi; Blankstein, Ron; Falk, Rodney H; Kwong, Raymond Y

2012-09-01

The aim of this study was to perform direct quantification of myocardial extracellular volume fraction (ECF) with T1-weighted cardiac magnetic resonance (CMR) imaging in patients suspected to have infiltrative heart disease. Infiltrative heart disease refers to accumulation of abnormal substances within the myocardium. Qualitative assessment of late gadolinium enhancement (LGE) remains the most commonly used method for CMR evaluation of patients suspected with myocardial infiltration. This technique is widely available and can be performed in a reproducible and standardized manner. However, the degree of extracellular matrix expansion due to myocardial infiltration in the intercellular space has, to date, not been amenable to noninvasive quantification with LGE. We performed 3-T CMR in 38 patients (mean age 68 ± 15 years) who were referred for assessment of infiltrative heart disease and also in 9 healthy volunteers as control subjects. The T1 quantification by Look-Locker gradient-echo before and after contrast determined segmental myocardial partition coefficients. The ECF was obtained by referencing the tissue partition coefficient for gadolinium to the plasma volume fraction in blood, derived from serum hematocrit. Cine CMR and LGE imaging in matching locations were also performed. Seventeen patients (45%) had cardiac amyloidosis (CA) (biopsy-confirmed or clinically highly probable), 20 (53%) had a non-amyloid cardiomyopathy, and 1 had lysosomal storage disease. Median global ECF was substantially higher in CA patients (0.49) compared with non-amyloid cardiomyopathy patients (0.33, p < 0.0001) and volunteers (0.24, p = 0.0001). The ECF strongly correlated with visually assessed segmental LGE (r = 0.80, p < 0.0001) and LV mass index (r = 0.69, p < 0.0001), reflecting severity of myocardial infiltration. In patients with CA, ECF was highest in segments with LGE, although it remained elevated in segments without qualitative LGE. The CMR ECF quantification

Adipose Tissue Quantification by Imaging Methods: A Proposed Classification

PubMed Central

Shen, Wei; Wang, ZiMian; Punyanita, Mark; Lei, Jianbo; Sinav, Ahmet; Kral, John G.; Imielinska, Celina; Ross, Robert; Heymsfield, Steven B.

2007-01-01

Recent advances in imaging techniques and understanding of differences in the molecular biology of adipose tissue has rendered classical anatomy obsolete, requiring a new classification of the topography of adipose tissue. Adipose tissue is one of the largest body compartments, yet a classification that defines specific adipose tissue depots based on their anatomic location and related functions is lacking. The absence of an accepted taxonomy poses problems for investigators studying adipose tissue topography and its functional correlates. The aim of this review was to critically examine the literature on imaging of whole body and regional adipose tissue and to create the first systematic classification of adipose tissue topography. Adipose tissue terminology was examined in over 100 original publications. Our analysis revealed inconsistencies in the use of specific definitions, especially for the compartment termed “visceral” adipose tissue. This analysis leads us to propose an updated classification of total body and regional adipose tissue, providing a well-defined basis for correlating imaging studies of specific adipose tissue depots with molecular processes. PMID:12529479

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

PubMed Central

2010-01-01

Background Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Results Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. Conclusions We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system. PMID:20492695

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples.

PubMed

Mehta, Rohini; Birerdinc, Aybike; Hossain, Noreen; Afendy, Arian; Chandhoke, Vikas; Younossi, Zobair; Baranova, Ancha

2010-05-21

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

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.

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

Colour thresholding and objective quantification in bioimaging

NASA Technical Reports Server (NTRS)

Fermin, C. D.; Gerber, M. A.; Torre-Bueno, J. R.

1992-01-01

Computer imaging is rapidly becoming an indispensable tool for the quantification of variables in research and medicine. Whilst its use in medicine has largely been limited to qualitative observations, imaging in applied basic sciences, medical research and biotechnology demands objective quantification of the variables in question. In black and white densitometry (0-256 levels of intensity) the separation of subtle differences between closely related hues from stains is sometimes very difficult. True-colour and real-time video microscopy analysis offer choices not previously available with monochrome systems. In this paper we demonstrate the usefulness of colour thresholding, which has so far proven indispensable for proper objective quantification of the products of histochemical reactions and/or subtle differences in tissue and cells. In addition, we provide interested, but untrained readers with basic information that may assist decisions regarding the most suitable set-up for a project under consideration. Data from projects in progress at Tulane are shown to illustrate the advantage of colour thresholding over monochrome densitometry and for objective quantification of subtle colour differences between experimental and control samples.

Quantification of petroleum-type hydrocarbons in avian tissue

USGS Publications Warehouse

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.

Histopathological Diagnostic Discrepancies in Soft Tissue Tumours Referred to a Specialist Centre: Reassessment in the Era of Ancillary Molecular Diagnosis

PubMed Central

Thway, Khin; Mubako, Taka

2014-01-01

Introduction. Soft tissue tumour pathology is a highly specialised area of surgical pathology, but soft tissue neoplasms can occur at virtually all sites and are therefore encountered by a wide population of surgical pathologists. Potential sarcomas require referral to specialist centres for review by pathologists who see a large number of soft tissue lesions and where appropriate ancillary investigations can be performed. We have previously assessed the types of diagnostic discrepancies between referring and final diagnosis for soft tissue lesions referred to our tertiary centre. We now reaudit this 6 years later, assessing changes in discrepancy patterns, particularly in relation to the now widespread use of ancillary molecular diagnostic techniques which were not prevalent in our original study. Materials and Methods. We compared the sarcoma unit's histopathology reports with referring reports on 348 specimens from 286 patients with suspected or proven soft tissue tumours in a one-year period. Results. Diagnostic agreement was seen in 250 cases (71.8%), with 57 (16.4%) major and 41 (11.8%) minor discrepancies. There were 23 cases of benign/malignant discrepancies (23.5% of all discrepancies). 50 ancillary molecular tests were performed, 33 for aiding diagnosis and 17 mutational analyses for gastrointestinal stromal tumour to guide therapy. Findings from ancillary techniques contributed to 3 major and 4 minor discrepancies. While the results were broadly similar to those of the previous study, there was an increase in frequency of major discrepancies. Conclusion. Six years following our previous study and notably now in an era of widespread ancillary molecular diagnosis, the overall discrepancy rate between referral and tertiary centre diagnosis remains similar, but there is an increase in frequency of major discrepancies likely to alter patient management. A possible reason for the increase in major discrepancies is the increasing lack of exposure to soft tissue

Histopathological diagnostic discrepancies in soft tissue tumours referred to a specialist centre: reassessment in the era of ancillary molecular diagnosis.

PubMed

Thway, Khin; Wang, Jayson; Mubako, Taka; Fisher, Cyril

2014-01-01

Introduction. Soft tissue tumour pathology is a highly specialised area of surgical pathology, but soft tissue neoplasms can occur at virtually all sites and are therefore encountered by a wide population of surgical pathologists. Potential sarcomas require referral to specialist centres for review by pathologists who see a large number of soft tissue lesions and where appropriate ancillary investigations can be performed. We have previously assessed the types of diagnostic discrepancies between referring and final diagnosis for soft tissue lesions referred to our tertiary centre. We now reaudit this 6 years later, assessing changes in discrepancy patterns, particularly in relation to the now widespread use of ancillary molecular diagnostic techniques which were not prevalent in our original study. Materials and Methods. We compared the sarcoma unit's histopathology reports with referring reports on 348 specimens from 286 patients with suspected or proven soft tissue tumours in a one-year period. Results. Diagnostic agreement was seen in 250 cases (71.8%), with 57 (16.4%) major and 41 (11.8%) minor discrepancies. There were 23 cases of benign/malignant discrepancies (23.5% of all discrepancies). 50 ancillary molecular tests were performed, 33 for aiding diagnosis and 17 mutational analyses for gastrointestinal stromal tumour to guide therapy. Findings from ancillary techniques contributed to 3 major and 4 minor discrepancies. While the results were broadly similar to those of the previous study, there was an increase in frequency of major discrepancies. Conclusion. Six years following our previous study and notably now in an era of widespread ancillary molecular diagnosis, the overall discrepancy rate between referral and tertiary centre diagnosis remains similar, but there is an increase in frequency of major discrepancies likely to alter patient management. A possible reason for the increase in major discrepancies is the increasing lack of exposure to soft tissue

Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing.

PubMed

Etich, Julia; Bergmeier, Vera; Pitzler, Lena; Brachvogel, Bent

2017-03-01

Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing. Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs. Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, RNU6B was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1β and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to Gapdh the normalization to multiple reference genes gave a similar outcome. RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.

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.

Photoacoustic bio-quantification of graphene based nanomaterials at a single cell level (Conference Presentation)

NASA Astrophysics Data System (ADS)

Nedosekin, Dmitry A.; Nolan, Jacqueline; Biris, Alexandru S.; Zharov, Vladimir P.

2017-03-01

Arkansas Nanomedicine Center at the University of Arkansas for Medical Sciences in collaboration with other Arkansas Universities and the FDA-based National Center of Toxicological Research in Jefferson, AR is developing novel techniques for rapid quantification of graphene-based nanomaterials (GBNs) in various biological samples. All-carbon GBNs have wide range of potential applications in industry, agriculture, food processing and medicine; however, quantification of GBNs is difficult in carbon reach biological tissues. The accurate quantification of GBNs is essential for research on material toxicity and the development of GBNs-based drug delivery platforms. We have developed microscopy and cytometry platforms for detection and quantification of GBNs in single cells, tissue and blood samples using photoacoustic contrast of GBNs. We demonstrated PA quantification of individual graphene uptake by single cells. High-resolution PA microscopy provided mapping of GBN distribution within live cells to establish correlation with intracellular toxic phenomena using apoptotic and necrotic assays. This new methodology and corresponding technical platform provide the insight on possible toxicological risks of GBNs at singe cells levels. In addition, in vivo PA image flow cytometry demonstrated the capability to monitor of GBNs pharmacokinetics in mouse model and to map the resulting biodistribution of GBNs in mouse tissues. The integrated PA platform provided an unprecedented sensitivity toward GBNs and allowed to enhance conventional toxicology research by providing a direct correlation between uptake of GBNs at a single cell level and cell viability status.

Array-based comparative genomic hybridization-guided identification of reference genes for normalization of real-time quantitative polymerase chain reaction assay data for lymphomas, histiocytic sarcomas, and osteosarcomas of dogs.

PubMed

Tsai, Pei-Chien; Breen, Matthew

2012-09-01

To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs. Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues. Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs. LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes. Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study.

PubMed

Durrenberger, Pascal F; Fernando, Francisca S; Magliozzi, Roberta; Kashefi, Samira N; Bonnert, Timothy P; Ferrer, Isidro; Seilhean, Danielle; Nait-Oumesmar, Brahim; Schmitt, Andrea; Gebicke-Haerter, Peter J; Falkai, Peter; Grünblatt, Edna; Palkovits, Miklos; Parchi, Piero; Capellari, Sabina; Arzberger, Thomas; Kretzschmar, Hans; Roncaroli, Federico; Dexter, David T; Reynolds, Richard

2012-12-01

The use of an appropriate reference gene to ensure accurate normalisation is crucial for the correct quantification of gene expression using qPCR assays and RNA arrays. The main criterion for a gene to qualify as a reference gene is a stable expression across various cell types and experimental settings. Several reference genes are commonly in use but more and more evidence reveals variations in their expression due to the presence of on-going neuropathological disease processes, raising doubts concerning their use. We conducted an analysis of genome-wide changes of gene expression in the human central nervous system (CNS) covering several neurological disorders and regions, including the spinal cord, and were able to identify a number of novel stable reference genes. We tested the stability of expression of eight novel (ATP5E, AARS, GAPVD1, CSNK2B, XPNPEP1, OSBP, NAT5 and DCTN2) and four more commonly used (BECN1, GAPDH, QARS and TUBB) reference genes in a smaller cohort using RT-qPCR. The most stable genes out of the 12 reference genes were tested as normaliser to validate increased levels of a target gene in CNS disease. We found that in human post-mortem tissue the novel reference genes, XPNPEP1 and AARS, were efficient in replicating microarray target gene expression levels and that XPNPEP1 was more efficient as a normaliser than BECN1, which has been shown to change in expression as a consequence of neuronal cell loss. We provide herein one more suitable novel reference gene, XPNPEP1, with no current neuroinflammatory or neurodegenerative associations that can be used for gene quantitative gene expression studies with human CNS post-mortem tissue and also suggest a list of potential other candidates. These data also emphasise the importance of organ/tissue-specific stably expressed genes as reference genes for RNA studies.

Use of internal references for assessing CT density measurements of the pelvis as replacement for use of an external phantom.

PubMed

Boomsma, Martijn F; Slouwerhof, Inge; van Dalen, Jorn A; Edens, Mireille A; Mueller, Dirk; Milles, Julien; Maas, Mario

2015-11-01

The purpose of this research is to study the use of an internal reference standard for fat- and muscle as a replacement for an external reference standard with a phantom. By using a phantomless internal reference standard, Hounsfield unit (HU) measurements of various tissues can potentially be assessed in patients with a CT scan of the pelvis without an added phantom at time of CT acquisition. This paves the way for development of a tool for quantification of the change in tissue density in one patient over time and between patients. This could make every CT scan made without contrast available for research purposes. Fifty patients with unilateral metal-on-metal total hip replacements, scanned together with a calibration reference phantom used in bone mineral density measurements, were included in this study. On computed tomography scans of the pelvis without the use of intravenous iodine contrast, reference values for fat and muscle were measured in the phantom as well as within the patient's body. The conformity between the references was examined with the intra-class correlation coefficient. The mean HU (± SD) of reference values for fat for the internal- and phantom references were -91.5 (±7.0) and -90.9 (±7.8), respectively. For muscle, the mean HU (± SD) for the internal- and phantom references were 59.2 (±6.2) and 60.0 (±7.2), respectively. The intra-class correlation coefficients for fat and muscle were 0.90 and 0.84 respectively and show excellent agreement between the phantom and internal references. Internal references can be used with similar accuracy as references from an external phantom. There is no need to use an external phantom to asses CT density measurements of body tissue.

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

Issues in quantification of registered respiratory gated PET/CT in the lung

NASA Astrophysics Data System (ADS)

Cuplov, Vesna; Holman, Beverley F.; McClelland, Jamie; Modat, Marc; Hutton, Brian F.; Thielemans, Kris

2018-01-01

PET/CT quantification of lung tissue is limited by several difficulties: the lung density and local volume changes during respiration, the anatomical mismatch between PET and CT and the relative contributions of tissue, air and blood to the PET signal (the tissue fraction effect). Air fraction correction (AFC) has been shown to improve PET image quantification in the lungs. Methods to correct for the movement and anatomical mismatch involve respiratory gating and image registration techniques. While conventional registration methods only account for spatial mismatch, the Jacobian determinant of the deformable registration transformation field can be used to estimate local volume changes and could therefore potentially be used to correct (i.e. Jacobian Correction, JC) the PET signal for changes in concentration due to local volume changes. This work aims to investigate the relationship between variations in the lung due to respiration, specifically density, tracer concentration and local volume changes. In particular, we study the effect of AFC and JC on PET quantitation after registration of respiratory gated PET/CT patient data. Six patients suffering from lung cancer with solitary pulmonary nodules underwent 18 F-FDG PET/cine-CT. The PET data were gated into six respiratory gates using displacement gating based on a real-time position management (RPM) signal and reconstructed with matched gated CT. The PET tracer concentration and tissue density were extracted from registered gated PET and CT images before and after corrections (AFC or JC) and compared to the values from the reference images. Before correction, we observed a linear correlation between the PET tracer concentration values and density. Across all gates and patients, the maximum relative change in PET tracer concentration before (after) AFC was found to be 16.2% (4.1%) and the maximum relative change in tissue density and PET tracer concentration before (after) JC was found to be 17.1% (5.5%) and 16

Quantification of confocal images of biofilms grown on irregular surfaces

PubMed Central

Ross, Stacy Sommerfeld; Tu, Mai Han; Falsetta, Megan L.; Ketterer, Margaret R.; Kiedrowski, Megan R.; Horswill, Alexander R.; Apicella, Michael A.; Reinhardt, Joseph M.; Fiegel, Jennifer

2014-01-01

Bacterial biofilms grow on many types of surfaces, including flat surfaces such as glass and metal and irregular surfaces such as rocks, biological tissues and polymers. While laser scanning confocal microscopy can provide high-resolution images of biofilms grown on any surface, quantification of biofilm-associated bacteria is currently limited to bacteria grown on flat surfaces. This can limit researchers studying irregular surfaces to qualitative analysis or quantification of only the total bacteria in an image. In this work, we introduce a new algorithm called modified connected volume filtration (MCVF) to quantify bacteria grown on top of an irregular surface that is fluorescently labeled or reflective. Using the MCVF algorithm, two new quantification parameters are introduced. The modified substratum coverage parameter enables quantification of the connected-biofilm bacteria on top of the surface and on the imaging substratum. The utility of MCVF and the modified substratum coverage parameter were shown with Pseudomonas aeruginosa and Staphylococcus aureus biofilms grown on human airway epithelial cells. A second parameter, the percent association, provides quantified data on the colocalization of the bacteria with a labeled component, including bacteria within a labeled tissue. The utility of quantifying the bacteria associated with the cell cytoplasm was demonstrated with Neisseria gonorrhoeae biofilms grown on cervical epithelial cells. This algorithm provides more flexibility and quantitative ability to researchers studying biofilms grown on a variety of irregular substrata. PMID:24632515

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.

Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

PubMed

Carraro, R; Dalla Rovere, G; Ferraresso, S; Carraro, L; Franch, R; Toffan, A; Pascoli, F; Patarnello, T; Bargelloni, L

2018-02-01

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease. © 2017 John Wiley & Sons Ltd.

Shear-wave elastography - virtual touch tissue quantification of fetal placentas with a single umbilical artery.

PubMed

Arslan, Harun; Tolunay, Harun Egemen; Cim, Numan; Boza, Barış; Yavuz, Alpaslan; İlik, İbrahim; Sahin, Hanim Guler; Yildizhan, Recep

2018-02-25

In this study, we aimed to evaluate the elasticities of fetal placentas with a single umbilical artery using the Virtual Touch Tissue Quantification (VTTQ) technique. Pregnant women with fetuses with a single umbilical artery (SUA) and pregnant women with fetuses having three vessel cord (3VC) at 18-22 weeks of gestation were enrolled in the research. The placentas were evaluated and divided into three equal parts as the inner 1/3 of the placenta (fetal edge), the outer 1/3 of the placenta (maternal edge) and the central 1/3 of the placenta (central part). Shear-wave velocity (SWV) measurements were used in the elastographic evaluation of placentas by VTTQ. Forty pregnant women were included in the study (n = 20 SUA, n = 20 three vessel cord pregnant women). The placental Acoustic Radiation Force Impulse (VTTQ) of the placenta regarding SWV measurement values of the fetal edge of the placenta in the fetuses with SUA and the control group were 0.876 and 0.957 m/sec, respectively. A significant statistical difference was found between the groups regarding the measurement of the stiffness of fetal placenta (p = 0.021). There was no significant difference between the measured stiffness values of the central or outer region of the placentas. In this study, we found lower SWV scores for the fetal edge of the placenta with SUA. This finding may reflect tissue elasticity level, and we hope that the use of the VTTQ technique may contribute to predicting the pregnancy-related morbidities of fetuses with SUA in the future.

Quantification of tissue oxygenation levels using diffuse reflectance spectroscopy

NASA Astrophysics Data System (ADS)

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

2011-08-01

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

Design and characterization of a direct ELISA for the detection and quantification of leucomalachite green

PubMed Central

Singh, Gurmit; Koerner, Terence; Gelinas, Jean-Marc; Abbott, Michael; Brady, Beth; Huet, Anne-Catherine; Charlier, Caroline; Delahaut, Philippe; Godefroy, Samuel Benrejeb

2011-01-01

Malachite green (MG), a member of the N-methylated triphenylmethane class of dyes, has long been used to control fungal and protozoan infections in fish. MG is easily absorbed by fish during waterborne exposure and is rapidly metabolized into leucomalachite green (LMG), which is known for its long residence time in edible fish tissue. This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of LMG in fish tissue. This development includes a simple and versatile method for the conversion of LMG to monodesmethyl-LMG, which is then conjugated to bovine serum albumin (BSA) to produce an immunogenic material. Rabbit polyclonal antibodies are generated against this immunogen, purified and used to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the screening and quantification of LMG in fish tissue. The assay performed well, with a limit of detection (LOD) and limit of quantification (LOQ) of 0.1 and 0.3 ng g−1 of fish tissue, respectively. The average extraction efficiency from a matrix of tilapia fillets was approximately 73% and the day-to-day reproducibility for these extractions in the assay was between 5 and 10%. PMID:21623496

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

PubMed Central

Maier, Helena J.; Van Borm, Steven; Young, John R.; Fife, Mark

2016-01-01

Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. PMID:27537060

Reference spectra from squamous epithelium and connective tissue allow whole section proteomics analysis.

PubMed

Roesch-Ely, Mariana; Schnölzer, Martina; Nees, Matthias; Plinkert, Peter K; Bosch, Franz X

2010-01-01

We reasoned that micro-dissection of tumour cells for protein expression studies should be omitted since tumour-stroma interactions are an important part of the biology of solid tumours. To study such interactions in head and neck squamous cell carcinoma (HNSCC) development, we generated reference protein spectra for normal squamous epithelium and connective tissue by SELDI-TOF-MS. Calgranulins A and B, Annexin1 and Histone H4 were found to be strongly enriched in the epithelium. The alpha-defensins 1-3 and the haemoglobin subunits were identified in the connective tissue. Tumour-distant epithelia, representing early pre-malignant lesions, showed up-regulated expression of the stromal alpha-defensins, whereas the epithelial Annexin 1 was down-regulated. Thus, tumour microenvironment interactions occur very early in the carcinogenic process. These data demonstrate that omitting micro-dissection is actually beneficial for studying changes in protein expression during development and progression of solid tumours.

Computerized whole slide quantification shows increased microvascular density in pT2 prostate cancer as compared to normal prostate tissue.

PubMed

van Niekerk, Cornelis G; van der Laak, Jeroen A W M; Börger, M Elisa; Huisman, Henk-Jan; Witjes, J Alfred; Barentsz, Jelle O; Hulsbergen-van de Kaa, Christina A

2009-01-01

Contrast enhanced imaging enables powerful, non-invasive diagnostics, important for detection and staging of early prostate cancer. The uptake of contrast agent is increased in prostate cancer as compared to normal prostate tissue. To reveal the underlying physiological mechanisms, quantification of tissue components in pathology specimens may yield important information. Aim of this study was to investigate whether microvascularity is increased in prostate confined cancer (pT2). Radical prostatectomy specimens of 26 patients were selected for organ confined peripheral zone tumors which were restricted to one side of the prostate. Microvessels were visualized by immunohistochemistry against CD31. Specimens were scanned using a computer controlled microscope and scanning stage and vessels were recognized automatically. Pseudocolor mappings were produced showing number of vascular profiles (MVD), vascular area (MVA) and perimeter (MVP) in an overview of the entire prostate transection. MVD is a common measure for vascularity, whereas MVA represents the 3D vascular volume and MVP the perfused surface area. Mean, coefficient of variation and 75th percentile of these parameters were calculated automatically in manually indicated areas, consisting of the entire tumor area and the corresponding normal area in the contra lateral side of the prostate. The mappings clearly indicate areas of increased vascularity in prostate transections. In tumor tissue an increase was found compared to normal tissue of 81%, 49%, and 62% for mean MVD, mean MVA and mean MVP, respectively (P < 0.001 for all comparisons). In contrast, the heterogeneity in tumor vasculature was significantly decreased as compared to normal prostate (P < 0.001). Characteristics of microvasculature deviated significantly in pT2 prostate tumor as compared to normal tissue. Copyright 2008 Wiley-Liss, Inc.

Recent application of quantification II in Japanese medical research.

PubMed Central

Suzuki, T; Kudo, A

1979-01-01

Hayashi's Quantification II is a method of multivariate discrimination analysis to manipulate attribute data as predictor variables. It is very useful in the medical research field for estimation, diagnosis, prognosis, evaluation of epidemiological factors, and other problems based on multiplicity of attribute data. In Japan, this method is so well known that most of the computer program packages include the Hayashi Quantification, but it seems to be yet unfamiliar with the method for researchers outside Japan. In view of this situation, we introduced 19 selected articles of recent applications of the Quantification II in Japanese medical research. In reviewing these papers, special mention is made to clarify how the researchers were satisfied with findings provided by the method. At the same time, some recommendations are made about terminology and program packages. Also a brief discussion of the background of the quantification methods is given with special reference to the Behaviormetric Society of Japan. PMID:540587

Colorimetric Quantification and in Situ Detection of Collagen

ERIC Educational Resources Information Center

Esteban, Francisco J.; del Moral, Maria L.; Sanchez-Lopez, Ana M.; Blanco, Santos; Jimenez, Ana; Hernandez, Raquel; Pedrosa, Juan A.; Peinado, Maria A.

2005-01-01

A simple multidisciplinary and inexpensive laboratory exercise is proposed, in which the undergraduate student may correlate biochemical and anatomical findings. The entire practical session can be completed in one 2.5-3 hour laboratory period, and consists of the quantification of collagen and total protein content from tissue sections--without…

Artifacts Quantification of Metal Implants in MRI

NASA Astrophysics Data System (ADS)

Vrachnis, I. N.; Vlachopoulos, G. F.; Maris, T. G.; Costaridou, L. I.

2017-11-01

The presence of materials with different magnetic properties, such as metal implants, causes distortion of the magnetic field locally, resulting in signal voids and pile ups, i.e. susceptibility artifacts in MRI. Quantitative and unbiased measurement of the artifact is prerequisite for optimization of acquisition parameters. In this study an image gradient based segmentation method is proposed for susceptibility artifact quantification. The method captures abrupt signal alterations by calculation of the image gradient. Then the artifact is quantified in terms of its extent by an automated cross entropy thresholding method as image area percentage. The proposed method for artifact quantification was tested in phantoms containing two orthopedic implants with significantly different magnetic permeabilities. The method was compared against a method proposed in the literature, considered as a reference, demonstrating moderate to good correlation (Spearman’s rho = 0.62 and 0.802 in case of titanium and stainless steel implants). The automated character of the proposed quantification method seems promising towards MRI acquisition parameter optimization.

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

PubMed

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

2017-01-24

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2014-10-01

Online and non-invasive quantification of critical tissue engineering (TE) construct quality attributes in TE bioreactors is indispensable for the cost-effective up-scaling and automation of cellular construct manufacturing. However, appropriate monitoring techniques for cellular constructs in bioreactors are still lacking. This study presents a generic and robust approach to determine cell number and metabolic activity of cell-based TE constructs in perfusion bioreactors based on single oxygen sensor data in dynamic perfusion conditions. A data-based mechanistic modeling technique was used that is able to correlate the number of cells within the scaffold (R(2)  = 0.80) and the metabolic activity of the cells (R(2)  = 0.82) to the dynamics of the oxygen response to step changes in the perfusion rate. This generic non-destructive measurement technique is effective for a large range of cells, from as low as 1.0 × 10(5) cells to potentially multiple millions of cells, and can open-up new possibilities for effective bioprocess monitoring. © 2014 Wiley Periodicals, Inc.

Technical evaluation of Virtual Touch™ tissue quantification and elastography in benign and malignant breast tumors

PubMed Central

JIANG, QUAN; ZHANG, YUAN; CHEN, JIAN; ZHANG, YUN-XIAO; HE, ZHU

2014-01-01

The aim of this study was to investigate the diagnostic value of the Virtual Touch™ tissue quantification (VTQ) and elastosonography technologies in benign and malignant breast tumors. Routine preoperative ultrasound, elastosonography and VTQ examinations were performed on 86 patients with breast lesions. The elastosonography score and VTQ speed grouping of each lesion were measured and compared with the pathological findings. The difference in the elastosonography score between the benign and malignant breast tumors was statistically significant (P<0.05). The detection rate for an elastosonography score of 1–3 points in benign tumors was 68.09% and that for an elastosonography score of 4–5 points in malignant tumors was 82.05%. The difference in VTQ speed values between the benign and malignant tumors was also statistically significant (P<0.05). In addition, the diagnostic accuracy of conventional ultrasound, elastosonography, VTQ technology and the combined methods showed statistically significant differences (P<0.05). The use of the three technologies in combination significantly improved the diagnostic accuracy to 91.86%. In conclusion, the combination of conventional ultrasound, elastosonography and VTQ technology can significantly improve accuracy in the diagnosis of breast cancer. PMID:25187797

Acoustic radiation force impulse imaging with Virtual Touch™ tissue quantification: mean shear wave velocity of malignant and benign breast masses.

PubMed

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

2013-01-01

Acoustic radiation force impulse imaging (ARFI) with Virtual Touch™ tissue quantification (VTTQ) enables the determination of shear wave velocity (SWV) in meters per second (m/s). The aim of our study was to describe the mean SWV in normal breast tissue and various breast masses. We performed measurements of SWV with ARFI VTTQ in 145 breast masses (57 malignant, 88 benign) and in the adjacent breast parenchyma and adipose tissue. The mean SWV as well as the rate of successful measurements were analyzed. The difference between adipose tissue and parenchyma was statistically significant (3.05 versus 3.65 m/s) (P < 0.001). Focusing on breast masses, numerous measurements exceeded the upper limit of possible measurement (≥9.10 m/s, indicated as "X.XX m/s"). Nevertheless, the difference between the malignant and benign masses was statistically significant (8.38 ± 1.99 m/s versus 5.39 ± 2.95 m/s) (P < 0.001). The best diagnostic accuracy (75.9%) was achieved when the cutoff point for malignancy was set to 9.10 m/s in ARFI VTTQ. This implies that the SWV was regarded as suspicious when the upper limit of possible measurement was exceeded and the machine returned the value X.XX m/s. In conclusion, ARFI VTTQ is a feasible method for measurement of SWV in a region of interest. Furthermore, we propose the event of a highly elevated SWV as a significant criterion for malignancy. However, the method is technically not yet fully developed, and the problem of unsuccessful measurements must still be solved.

Recurrence quantification as potential bio-markers for diagnosis of pre-cancer

NASA Astrophysics Data System (ADS)

Mukhopadhyay, Sabyasachi; Pratiher, Sawon; Barman, Ritwik; Pratiher, Souvik; Pradhan, Asima; Ghosh, Nirmalya; Panigrahi, Prasanta K.

2017-03-01

In this paper, the spectroscopy signals have been analyzed in recurrence plots (RP), and extract recurrence quantification analysis (RQA) parameters from the RP in order to classify the tissues into normal and different precancerous grades. Three RQA parameters have been quantified in order to extract the important features in the spectroscopy data. These features have been fed to different classifiers for classification. Simulation results validate the efficacy of the recurrence quantification as potential bio-markers for diagnosis of pre-cancer.

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.

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

PubMed Central

Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

2006-01-01

Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary

Revisiting the Logan plot to account for non-negligible blood volume in brain tissue.

PubMed

Schain, Martin; Fazio, Patrik; Mrzljak, Ladislav; Amini, Nahid; Al-Tawil, Nabil; Fitzer-Attas, Cheryl; Bronzova, Juliana; Landwehrmeyer, Bernhard; Sampaio, Christina; Halldin, Christer; Varrone, Andrea

2017-08-18

Reference tissue-based quantification of brain PET data does not typically include correction for signal originating from blood vessels, which is known to result in biased outcome measures. The bias extent depends on the amount of radioactivity in the blood vessels. In this study, we seek to revisit the well-established Logan plot and derive alternative formulations that provide estimation of distribution volume ratios (DVRs) that are corrected for the signal originating from the vasculature. New expressions for the Logan plot based on arterial input function and reference tissue were derived, which included explicit terms for whole blood radioactivity. The new methods were evaluated using PET data acquired using [ 11 C]raclopride and [ 18 F]MNI-659. The two-tissue compartment model (2TCM), with which signal originating from blood can be explicitly modeled, was used as a gold standard. DVR values obtained for [ 11 C]raclopride using the either blood-based or reference tissue-based Logan plot were systematically underestimated compared to 2TCM, and for [ 18 F]MNI-659, a proportionality bias was observed, i.e., the bias varied across regions. The biases disappeared when optimal blood-signal correction was used for respective tracer, although for the case of [ 18 F]MNI-659 a small but systematic overestimation of DVR was still observed. The new method appears to remove the bias introduced due to absence of correction for blood volume in regular graphical analysis and can be considered in clinical studies. Further studies are however required to derive a generic mapping between plasma and whole-blood radioactivity levels.

Equilibrium Passive Sampling of POP in Lipid-Rich and Lean Fish Tissue: Quality Control Using Performance Reference Compounds.

PubMed

Rusina, Tatsiana P; Carlsson, Pernilla; Vrana, Branislav; Smedes, Foppe

2017-10-03

Passive sampling is widely used to measure levels of contaminants in various environmental matrices, including fish tissue. Equilibrium passive sampling (EPS) of persistent organic pollutants (POP) in fish tissue has been hitherto limited to application in lipid-rich tissue. We tested several exposure methods to extend EPS applicability to lean tissue. Thin-film polydimethylsiloxane (PDMS) passive samplers were exposed statically to intact fillet and fish homogenate and dynamically by rolling with cut fillet cubes. The release of performance reference compounds (PRC) dosed to passive samplers prior to exposure was used to monitor the exchange process. The sampler-tissue exchange was isotropic, and PRC were shown to be good indicators of sampler-tissue equilibration status. The dynamic exposures demonstrated equilibrium attainment in less than 2 days for all three tested fish species, including lean fish containing 1% lipid. Lipid-based concentrations derived from EPS were in good agreement with lipid-normalized concentrations obtained using conventional solvent extraction. The developed in-tissue EPS method is robust and has potential for application in chemical monitoring of biota and bioaccumulation studies.

Referred pain and cutaneous responses from deep tissue electrical pain stimulation in the groin.

PubMed

Aasvang, E K; Werner, M U; Kehlet, H

2015-08-01

Persistent postherniotomy pain is located around the scar and external inguinal ring and is often described as deep rather than cutaneous, with frequent complaints of pain in adjacent areas. Whether this pain is due to local pathology or referred/projected pain is unknown, hindering mechanism-based treatment. Deep tissue electrical pain stimulation by needle electrodes in the right groin (rectus muscle, ilioinguinal/iliohypogastric nerve and perispermatic cord) was combined with assessment of referred/projected pain and the cutaneous heat pain threshold (HPT) at three prespecified areas (both groins and the lower right arm) in 19 healthy subjects. The assessment was repeated 10 days later to assess the reproducibility of individual responses. Deep electrical stimulation elicited pain at the stimulation site in all subjects, and in 15 subjects, pain from areas outside the stimulation area was reported, with 90-100% having the same response on both days, depending on the location. Deep pain stimulation significantly increased the cutaneous HPT (P<0.014). Individual HPT responses before and during deep electrical pain stimulation were significantly correlated (ρ>0.474, P≤0.040) at the two test days for the majority of test areas. Our results corroborate a systematic relationship between deep pain and changes in cutaneous nociception. The individual referred/projected pain patterns and cutaneous responses are variable, but reproducible, supporting individual differences in anatomy and sensory processing. Future studies investigating the responses to deep tissue electrical stimulation in persistent postherniotomy pain patients may advance our understanding of underlying pathophysiological mechanisms and strategies for treatment and prevention. ClinicalTrials.gov (NCT01701427). © The Author 2015. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Development of potential candidate reference materials for drugs in bottom sediment, cod and herring tissues.

PubMed

Baranowska, Irena; Buszewski, Bogusław; Namieśnik, Jacek; Konieczka, Piotr; Magiera, Sylwia; Polkowska-Motrenko, Halina; Kościelniak, Paweł; Gadzała-Kopciuch, Renata; Woźniakiewicz, Aneta; Samczyński, Zbigniew; Kochańska, Kinga; Rutkowska, Małgorzata

2017-02-01

Regular use of a reference material and participation in a proficiency testing program can improve the reliability of analytical data. This paper presents the preparation of candidate reference materials for the drugs metoprolol, propranolol, carbamazepine, naproxen, and acenocoumarol in freshwater bottom sediment and cod and herring tissues. These reference materials are not available commercially. Drugs (between 7 ng/g and 32 ng/g) were added to the samples, and the spiked samples were freeze-dried, pulverized, sieved, homogenized, bottled, and sterilized by γ-irradiation to prepare the candidate materials. Procedures for extraction and liquid chromatography coupled with tandem mass spectrometry were developed to determine the drugs of interest in the studied material. Each target drug was quantified using two analytical procedures, and the results obtained from these two procedures were in good agreement with each other. Stability and homogeneity assessments were performed, and the relative uncertainties due to instability (for an expiration date of 12 months) and inhomogeneity were 10-25% and 4.0-6.8%, respectively. These procedures will be useful in the future production of reference materials. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Issues in quantification of registered respiratory gated PET/CT in the lung.

PubMed

Cuplov, Vesna; Holman, Beverley F; McClelland, Jamie; Modat, Marc; Hutton, Brian F; Thielemans, Kris

2017-12-14

PET/CT quantification of lung tissue is limited by several difficulties: the lung density and local volume changes during respiration, the anatomical mismatch between PET and CT and the relative contributions of tissue, air and blood to the PET signal (the tissue fraction effect). Air fraction correction (AFC) has been shown to improve PET image quantification in the lungs. Methods to correct for the movement and anatomical mismatch involve respiratory gating and image registration techniques. While conventional registration methods only account for spatial mismatch, the Jacobian determinant of the deformable registration transformation field can be used to estimate local volume changes and could therefore potentially be used to correct (i.e. Jacobian Correction, JC) the PET signal for changes in concentration due to local volume changes. This work aims to investigate the relationship between variations in the lung due to respiration, specifically density, tracer concentration and local volume changes. In particular, we study the effect of AFC and JC on PET quantitation after registration of respiratory gated PET/CT patient data. Six patients suffering from lung cancer with solitary pulmonary nodules underwent [Formula: see text]F-FDG PET/cine-CT. The PET data were gated into six respiratory gates using displacement gating based on a real-time position management (RPM) signal and reconstructed with matched gated CT. The PET tracer concentration and tissue density were extracted from registered gated PET and CT images before and after corrections (AFC or JC) and compared to the values from the reference images. Before correction, we observed a linear correlation between the PET tracer concentration values and density. Across all gates and patients, the maximum relative change in PET tracer concentration before (after) AFC was found to be 16.2% (4.1%) and the maximum relative change in tissue density and PET tracer concentration before (after) JC was found to be 17

Quantification of structural changes in acute inflammation by fractal dimension, angular second moment and correlation.

PubMed

Stankovic, Marija; Pantic, Igor; De Luka, Silvio R; Puskas, Nela; Zaletel, Ivan; Milutinovic-Smiljanic, Sanja; Pantic, Senka; Trbovich, Alexander M

2016-03-01

The aim of the study was to examine alteration and possible application of fractal dimension, angular second moment, and correlation for quantification of structural changes in acutely inflamed tissue. Acute inflammation was induced by injection of turpentine oil into the right and left hind limb muscles of mice, whereas control animals received intramuscular saline injection. After 12 h, animals were anesthetised and treated muscles collected. The tissue was stained by hematoxylin and eosin, digital micrographs produced, enabling determination of fractal dimension of the cells, angular second moment and correlation of studied tissue. Histopathological analysis showed presence of inflammatory infiltrate and tissue damage in inflammatory group, whereas tissue structure in control group was preserved, devoid of inflammatory infiltrate. Fractal dimension of the cells, angular second moment and correlation of treated tissue in inflammatory group decreased in comparison to the control group. In this study, we were first to observe and report that fractal dimension of the cells, angular second moment, and correlation were reduced in acutely inflamed tissue, indicating loss of overall complexity of the cells in the tissue, the tissue uniformity and structure regularity. Fractal dimension, angular second moment and correlation could be useful methods for quantification of structural changes in acute inflammation. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Tissue-specific selection of stable reference genes for real-time PCR normalization in an obese rat model.

PubMed

Cabiati, Manuela; Raucci, Serena; Caselli, Chiara; Guzzardi, Maria Angela; D'Amico, Andrea; Prescimone, Tommaso; Giannessi, Daniela; Del Ry, Silvia

2012-06-01

Obesity is a complex pathology with interacting and confounding causes due to the environment, hormonal signaling patterns, and genetic predisposition. At present, the Zucker rat is an eligible genetic model for research on obesity and metabolic syndrome, allowing scrutiny of gene expression profiles. Real-time PCR is the benchmark method for measuring mRNA expressions, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. In the Zucker rat model, no specific reference genes have been identified in myocardium, kidney, and lung, the main organs involved in this syndrome. The aim of this study was to select among ten candidates (Actb, Gapdh, Polr2a, Ywhag, Rpl13a, Sdha, Ppia, Tbp, Hprt1 and Tfrc) a set of reference genes that can be used for the normalization of mRNA expression data obtained by real-time PCR in obese and lean Zucker rats both at fasting and during acute hyperglycemia. The most stable genes in the heart were Sdha, Tbp, and Hprt1; in kidney, Tbp, Actb, and Gapdh were chosen, while Actb, Ywhag, and Sdha were selected as the most stably expressed set for pulmonary tissue. The normalization strategy was used to analyze mRNA expression of tumor necrosis factor α, the main inflammatory mediator in obesity, whose variations were more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance of having three stably expressed reference gene sets for use in the cardiac, renal, and pulmonary tissues of an experimental model of obese and hyperglycemic Zucker rats.

Fully automated gynecomastia quantification from low-dose chest CT

NASA Astrophysics Data System (ADS)

Liu, Shuang; Sonnenblick, Emily B.; Azour, Lea; Yankelevitz, David F.; Henschke, Claudia I.; Reeves, Anthony P.

2018-02-01

Gynecomastia is characterized by the enlargement of male breasts, which is a common and sometimes distressing condition found in over half of adult men over the age of 44. Although the majority of gynecomastia is physiologic or idiopathic, its occurrence may also associate with an extensive variety of underlying systemic disease or drug toxicity. With the recent large-scale implementation of annual lung cancer screening using low-dose chest CT (LDCT), gynecomastia is believed to be a frequent incidental finding on LDCT. A fully automated system for gynecomastia quantification from LDCT is presented in this paper. The whole breast region is first segmented using an anatomyorientated approach based on the propagation of pectoral muscle fronts in the vertical direction. The subareolar region is then localized, and the fibroglandular tissue within it is measured for the assessment of gynecomastia. The presented system was validated using 454 breast regions from non-contrast LDCT scans of 227 adult men. The ground truth was established by an experienced radiologist by classifying each breast into one of the five categorical scores. The automated measurements have been demonstrated to achieve promising performance for the gynecomastia diagnosis with the AUC of 0.86 for the ROC curve and have statistically significant Spearman correlation r=0.70 (p < 0.001) with the reference categorical grades. The encouraging results demonstrate the feasibility of fully automated gynecomastia quantification from LDCT, which may aid the early detection as well as the treatment of both gynecomastia and the underlying medical problems, if any, that cause gynecomastia.

Comparison of tissue processing methods for microvascular visualization in axolotls.

PubMed

Montoro, Rodrigo; Dickie, Renee

2017-01-01

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

Quantification of Dynamic 11C-Phenytoin PET Studies.

PubMed

Mansor, Syahir; Boellaard, Ronald; Froklage, Femke E; Bakker, Esther D M; Yaqub, Maqsood; Voskuyl, Rob A; Schwarte, Lothar A; Verbeek, Joost; Windhorst, Albert D; Lammertsma, Adriaan

2015-09-01

The overexpression of P-glycoprotein (Pgp) is thought to be an important mechanism of pharmacoresistance in epilepsy. Recently, (11)C-phenytoin has been evaluated preclinically as a tracer for Pgp. The aim of the present study was to assess the optimal plasma kinetic model for quantification of (11)C-phenytoin studies in humans. Dynamic (11)C-phenytoin PET scans of 6 healthy volunteers with arterial sampling were acquired twice on the same day and analyzed using single- and 2-tissue-compartment models with and without a blood volume parameter. Global and regional test-retest (TRT) variability was determined for both plasma to tissue rate constant (K1) and volume of distribution (VT). According to the Akaike information criterion, the reversible single-tissue-compartment model with blood volume parameter was the preferred plasma input model. Mean TRT variability ranged from 1.5% to 16.9% for K1 and from 0.5% to 5.8% for VT. Larger volumes of interest showed better repeatabilities than smaller regions. A 45-min scan provided essentially the same K1 and VT values as a 60-min scan. A reversible single-tissue-compartment model with blood volume seems to be a good candidate model for quantification of dynamic (11)C-phenytoin studies. Scan duration may be reduced to 45 min without notable loss of accuracy and precision of both K1 and VT, although this still needs to be confirmed under pathologic conditions. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

The Transcriptome of the Reference Potato Genome Solanum tuberosum Group Phureja Clone DM1-3 516R44

PubMed Central

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

2011-01-01

Advances in molecular breeding in potato have been limited by its complex biological system, which includes vegetative propagation, autotetraploidy, and extreme heterozygosity. The availability of the potato genome and accompanying gene complement with corresponding gene structure, location, and functional annotation are powerful resources for understanding this complex plant and advancing molecular breeding efforts. Here, we report a reference for the potato transcriptome using 32 tissues and growth conditions from the doubled monoploid Solanum tuberosum Group Phureja clone DM1-3 516R44 for which a genome sequence is available. Analysis of greater than 550 million RNA-Seq reads permitted the detection and quantification of expression levels of over 22,000 genes. Hierarchical clustering and principal component analyses captured the biological variability that accounts for gene expression differences among tissues suggesting tissue-specific gene expression, and genes with tissue or condition restricted expression. Using gene co-expression network analysis, we identified 18 gene modules that represent tissue-specific transcriptional networks of major potato organs and developmental stages. This information provides a powerful resource for potato research as well as studies on other members of the Solanaceae family. PMID:22046362

Quantification of meat proportions by measuring DNA contents in raw and boiled sausages using matrix-adapted calibrators and multiplex real-time PCR.

PubMed

Köppel, René; Eugster, Albert; Ruf, Jürg; Rentsch, Jürg

2012-01-01

The quantification of meat proportions in raw and boiled sausage according to the recipe was evaluated using three different calibrators. To measure the DNA contents from beef, pork, sheep (mutton), and horse, a tetraplex real-time PCR method was applied. Nineteen laboratories analyzed four meat products each made of different proportions of beef, pork, sheep, and horse meat. Three kinds of calibrators were used: raw and boiled sausages of known proportions ranging from 1 to 55% of meat, and a dilution series of DNA from muscle tissue. In general, results generated using calibration sausages were more accurate than those resulting from the use of DNA from muscle tissue, and exhibited smaller measurement uncertainties. Although differences between uses of raw and boiled calibration sausages were small, the most precise and accurate results were obtained by calibration with fine-textured boiled reference sausages.

Microfluidic extraction and microarray detection of biomarkers from cancer tissue slides

NASA Astrophysics Data System (ADS)

Nguyen, H. T.; Dupont, L. N.; Jean, A. M.; Géhin, T.; Chevolot, Y.; Laurenceau, E.; Gijs, M. A. M.

2018-03-01

We report here a new microfluidic method allowing for the quantification of human epidermal growth factor receptor 2 (HER2) expression levels from formalin-fixed breast cancer tissues. After partial extraction of proteins from the tissue slide, the extract is routed to an antibody (Ab) microarray for HER2 titration by fluorescence. Then the HER2-expressing cell area is evaluated by immunofluorescence (IF) staining of the tissue slide and used to normalize the fluorescent HER2 signal measured from the Ab microarray. The number of HER2 gene copies measured by fluorescence in situ hybridization (FISH) on an adjacent tissue slide is concordant with the normalized HER2 expression signal. This work is the first study implementing biomarker extraction and detection from cancer tissue slides using microfluidics in combination with a microarray system, paving the way for further developments towards multiplex and precise quantification of cancer biomarkers.

Optical probe with reference fiber

DOEpatents

Da Silva, Luiz B [Danville, CA; Chase, Charles L [Dublin, CA

2006-03-14

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

qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

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

Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch; Gallati, Sabina, E-mail: sabina.gallati@insel.ch; Schaller, Andre, E-mail: andre.schaller@insel.ch

2012-07-06

Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serialmore » qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct

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

PubMed

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

2015-01-01

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

Trace element reference values in tissues from inhabitants of the EU. XII. Development of BioReVa program for statistical treatment.

PubMed

Iversen, B S; Sabbioni, E; Fortaner, S; Pietra, R; Nicolotti, A

2003-01-20

Statistical data treatment is a key point in the assessment of trace element reference values being the conclusive stage of a comprehensive and organized evaluation process of metal concentration in human body fluids. The EURO TERVIHT project (Trace Elements Reference Values in Human Tissues) was started for evaluating, checking and suggesting harmonized procedures for the establishment of trace element reference intervals in body fluids and tissues. Unfortunately, different statistical approaches are being used in this research field making data comparison difficult and in some cases impossible. Although international organizations such as International Federation of Clinical Chemistry (IFCC) or International Union of Pure and Applied Chemistry (IUPAC) have issued recommended guidelines for reference values assessment, including the statistical data treatment, a unique format and a standardized data layout is still missing. The aim of the present study is to present a software (BioReVa) running under Microsoft Windows platform suitable for calculating the reference intervals of trace elements in body matrices. The main scope for creating an ease-of-use application was to control the data distribution, to establish the reference intervals according to the accepted recommendation, on the base of the simple statistic, to get a standard presentation of experimental data and to have an application to which further need could be integrated in future. BioReVa calculates the IFCC reference intervals as well as the coverage intervals recommended by IUPAC as a supplement to the IFCC intervals. Examples of reference values and reference intervals calculated with BioReVa software concern Pb and Se in blood; Cd, In and Cr in urine, Hg and Mo in hair of different general European populations. University of Michigan

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death Events and Fibrosis in Tissue Sections

PubMed Central

Krajewska, Maryla; Smith, Layton H.; Rong, Juan; Huang, Xianshu; Hyer, Marc L.; Zeps, Nikolajs; Iacopetta, Barry; Linke, Steven P.; Olson, Allen H.; Reed, John C.; Krajewski, Stan

2009-01-01

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections. (J Histochem Cytochem 57:649–663, 2009) PMID:19289554

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.

Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events.

PubMed

Pi, Liqun; Li, Xiang; Cao, Yiwei; Wang, Canhua; Pan, Liangwen; Yang, Litao

2015-04-01

Reference materials are important in accurate analysis of genetically modified organism (GMO) contents in food/feeds, and development of novel reference plasmid is a new trend in the research of GMO reference materials. Herein, we constructed a novel multi-targeting plasmid, pSOY, which contained seven event-specific sequences of five GM soybeans (MON89788-5', A2704-12-3', A5547-127-3', DP356043-5', DP305423-3', A2704-12-5', and A5547-127-5') and sequence of soybean endogenous reference gene Lectin. We evaluated the specificity, limit of detection and quantification, and applicability of pSOY in both qualitative and quantitative PCR analyses. The limit of detection (LOD) was as low as 20 copies in qualitative PCR, and the limit of quantification (LOQ) in quantitative PCR was 10 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and Lectin assays were higher than 90%, and the squared regression coefficients (R(2)) were more than 0.999. The quantification bias varied from 0.21% to 19.29%, and the relative standard deviations were from 1.08% to 9.84% in simulated samples analysis. All the results demonstrated that the developed multi-targeting plasmid, pSOY, was a credible substitute of matrix reference materials, and could be used as a reliable reference calibrator in the identification and quantification of multiple GM soybean events.

Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event.

PubMed

Weighardt, Florian; Barbati, Cristina; Paoletti, Claudia; Querci, Maddalena; Kay, Simon; De Beuckeleer, Marc; Van den Eede, Guy

2004-01-01

In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement

Absolute quantification methods in tissue near-infrared spectroscopy

NASA Astrophysics Data System (ADS)

Matcher, Steven J.; Kirkpatrick, Peter J.; Nahid, K.; Cope, Mark; Delpy, David T.

1995-05-01

Recent work aimed at providing an absolute measurement of tissue haemoglobin saturation and a new instrument development, the spatially resolved spectrometer (SRS), are discussed. The theoretical basis of operation of this device and its hardware implementation are described and the results of validation studies on tissue simulating phantoms are presented as are preliminary measurements on human volunteers and observations on patients undergoing neurosurgery. In its present form the instrument appears to produce absolute haemoglobin saturation values for resting human skeletal muscle and the normally perfused human head which are rather low based on physiological expectations. However, we obtained a tight correlation between the saturation values measured by the SRS instrument and those obtained from blood-gas analysis of samples drawn from a jugular bulb catheter in one neurosurgery subject during clamping of the right carotid arteries.

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

2016-05-10

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.

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

PubMed Central

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

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.

Total iodine quantification in fluids and tissues from iodine- or iodide-supplemented rats by ion chromatography following microwave-assisted digestion.

PubMed

Delgado, Guadalupe; Muñoz-Torres, Carolina; Orozco-Esquivel, Teresa; Anguiano, Brenda; Aceves, Carmen

2015-03-01

Iodine is a crucial component of thyroid hormones, and several reports have shown that iodine per se is implicated in the physiopathology of other organs. Innovative ion chromatography detection following a four-step temperature ramp microwave digestion in 25-50 mM nitric acid was developed to measure total iodine in biological fluids and tissue samples from female Sprague-Dawley rats supplemented with 0.05% molecular iodine (I2) or 0.05% potassium iodide (I(-)) in drinking water. The reported method allows the measurement of total iodine with a limit of quantification of 13.7 μg L(-1), recoveries of 96.3-100.3%, and intra- and inter-assay variations, of 3.5% and 7.4% respectively. Analysis of biological fluids showed that after 48 hours, iodine-supplemented animals exhibited significantly higher levels of total iodine in both serum and urine compared with those supplemented with iodide. The half-life of iodine in serum and urine measured over the first 48 h showed similar patterns for both the I2 (7.89 and 7.76 hours) and I(-) (8.27 and 8.90 hours) supplements. Differential uptake patterns were observed in tissues after 6 days of supplements, with I(-) preferentially retained by thyroid, lactating mammary gland, and milk, and a slightly but significantly higher capture of I2 in pituitary, ovary, and virgin mammary gland. We developed a rapid, selective, and accurate digestion method to process fluid and tissue samples that permits reproducible measurements of total iodine by ion chromatography; iodine or iodide supplement show a similar serum and urine half-life, but organ-specific uptake depends on the chemical form of the iodine supplement.

Normal Databases for the Relative Quantification of Myocardial Perfusion

PubMed Central

Rubeaux, Mathieu; Xu, Yuan; Germano, Guido; Berman, Daniel S.; Slomka, Piotr J.

2016-01-01

Purpose of review Myocardial perfusion imaging (MPI) with SPECT is performed clinically worldwide to detect and monitor coronary artery disease (CAD). MPI allows an objective quantification of myocardial perfusion at stress and rest. This established technique relies on normal databases to compare patient scans against reference normal limits. In this review, we aim to introduce the process of MPI quantification with normal databases and describe the associated perfusion quantitative measures that are used. Recent findings New equipment and new software reconstruction algorithms have been introduced which require the development of new normal limits. The appearance and regional count variations of normal MPI scan may differ between these new scanners and standard Anger cameras. Therefore, these new systems may require the determination of new normal limits to achieve optimal accuracy in relative myocardial perfusion quantification. Accurate diagnostic and prognostic results rivaling those obtained by expert readers can be obtained by this widely used technique. Summary Throughout this review, we emphasize the importance of the different normal databases and the need for specific databases relative to distinct imaging procedures. use of appropriate normal limits allows optimal quantification of MPI by taking into account subtle image differences due to the hardware and software used, and the population studied. PMID:28138354

Gaussian signal relaxation around spin echoes: Implications for precise reversible transverse relaxation quantification of pulmonary tissue at 1.5 and 3 Tesla.

PubMed

Zapp, Jascha; Domsch, Sebastian; Weingärtner, Sebastian; Schad, Lothar R

2017-05-01

To characterize the reversible transverse relaxation in pulmonary tissue and to study the benefit of a quadratic exponential (Gaussian) model over the commonly used linear exponential model for increased quantification precision. A point-resolved spectroscopy sequence was used for comprehensive sampling of the relaxation around spin echoes. Measurements were performed in an ex vivo tissue sample and in healthy volunteers at 1.5 Tesla (T) and 3 T. The goodness of fit using χred2 and the precision of the fitted relaxation time by means of its confidence interval were compared between the two relaxation models. The Gaussian model provides enhanced descriptions of pulmonary relaxation with lower χred2 by average factors of 4 ex vivo and 3 in volunteers. The Gaussian model indicates higher sensitivity to tissue structure alteration with increased precision of reversible transverse relaxation time measurements also by average factors of 4 ex vivo and 3 in volunteers. The mean relaxation times of the Gaussian model in volunteers are T2,G' = (1.97 ± 0.27) msec at 1.5 T and T2,G' = (0.83 ± 0.21) msec at 3 T. Pulmonary signal relaxation was found to be accurately modeled as Gaussian, providing a potential biomarker T2,G' with high sensitivity. Magn Reson Med 77:1938-1945, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

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

Determination of the purity of pharmaceutical reference materials by 1H NMR using the standardless PULCON methodology.

PubMed

Monakhova, Yulia B; Kohl-Himmelseher, Matthias; Kuballa, Thomas; Lachenmeier, Dirk W

2014-11-01

A fast and reliable nuclear magnetic resonance spectroscopic method for quantitative determination (qNMR) of targeted molecules in reference materials has been established using the ERETIC2 methodology (electronic reference to access in vivo concentrations) based on the PULCON principle (pulse length based concentration determination). The developed approach was validated for the analysis of pharmaceutical samples in the context of official medicines control, including ibandronic acid, amantadine, ambroxol and lercanidipine. The PULCON recoveries were above 94.3% and coefficients of variation (CVs) obtained by quantification of different targeted resonances ranged between 0.7% and 2.8%, demonstrating that the qNMR method is a precise tool for rapid quantification (approximately 15min) of reference materials and medicinal products. Generally, the values were within specification (certified values) provided by the manufactures. The results were in agreement with NMR quantification using an internal standard and validated reference HPLC analysis. The PULCON method was found to be a practical alternative with competitive precision and accuracy to the classical internal reference method and it proved to be applicable to different solvent conditions. The method can be recommended for routine use in medicines control laboratories, especially when the availability and costs of reference compounds are problematic. Copyright © 2014 Elsevier B.V. All rights reserved.

Microfabricated tissues for investigating traction forces involved in cell migration and tissue morphogenesis

PubMed Central

Nerger, Bryan A.; Siedlik, Michael J.; Nelson, Celeste M.

2016-01-01

Cell-generated forces drive an array of biological processes ranging from wound healing to tumor metastasis. Whereas experimental techniques such as traction force microscopy are capable of quantifying traction forces in multidimensional systems, the physical mechanisms by which these forces induce changes in tissue form remain to be elucidated. Understanding these mechanisms will ultimately require techniques that are capable of quantifying traction forces with high precision and accuracy in vivo or in systems that recapitulate in vivo conditions, such as microfabricated tissues and engineered substrata. To that end, here we review the fundamentals of traction forces, their quantification, and the use of microfabricated tissues designed to study these forces during cell migration and tissue morphogenesis. We emphasize the differences between traction forces in two- and three-dimensional systems, and highlight recently developed techniques for quantifying traction forces. PMID:28008471

Direct qPCR quantification using the Quantifiler(®) Trio DNA quantification kit.

PubMed

Liu, Jason Yingjie

2014-11-01

The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler(®) Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both low-level touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler(®) Trio kit enabled the detection of less than 10pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler(®) Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler(®) Trio kit) and the direct STR results (GlobalFiler™ PCR amplification kit(*)) for low-level touch DNA samples indicates that direct quantification using the Quantifiler(®) Trio DNA quantification kit is more reliable than the Quantifiler(®) Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10pg of DNA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Round robin test on quantification of amyloid-β 1-42 in cerebrospinal fluid by mass spectrometry.

PubMed

Pannee, Josef; Gobom, Johan; Shaw, Leslie M; Korecka, Magdalena; Chambers, Erin E; Lame, Mary; Jenkins, Rand; Mylott, William; Carrillo, Maria C; Zegers, Ingrid; Zetterberg, Henrik; Blennow, Kaj; Portelius, Erik

2016-01-01

Cerebrospinal fluid (CSF) amyloid-β 1-42 (Aβ42) is an important biomarker for Alzheimer's disease, both in diagnostics and to monitor disease-modifying therapies. However, there is a great need for standardization of methods used for quantification. To overcome problems associated with immunoassays, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a critical orthogonal alternative. We compared results for CSF Aβ42 quantification in a round robin study performed in four laboratories using similar sample preparation methods and LC-MS instrumentation. The LC-MS results showed excellent correlation between laboratories (r(2) >0.98), high analytical precision, and good correlation with enzyme-linked immunosorbent assay (r(2) >0.85). The use of a common reference sample further decreased interlaboratory variation. Our results indicate that LC-MS is suitable for absolute quantification of Aβ42 in CSF and highlight the importance of developing a certified reference material. Copyright © 2016 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

Quantification of Neural Ethanol and Acetaldehyde Using Headspace GC-MS

PubMed Central

Heit, Claire; Eriksson, Peter; Thompson, David C; Fritz, Kristofer S; Vasiliou, Vasilis

2016-01-01

BACKGROUND There is controversy regarding the active agent responsible for alcohol addiction. The theory that ethanol itself was the agent in alcohol drinking behavior was widely accepted until acetaldehyde was found in the brain. The importance of acetaldehyde formation in the brain role is still subject to speculation due to the lack of a method to accurately assay the acetaldehyde levels directly. A highly sensitive GC-MS method to reliably determine acetaldehyde concentration with certainty is needed to address whether neural acetaldehyde is indeed responsible for increased alcohol consumption. METHODS A headspace gas chromatograph coupled to selected ion monitoring mass spectrometry was utilized to develop a quantitative assay for acetaldehyde and ethanol. Our GC-MS approach was carried out using a Bruker Scion 436-GC SQ MS. RESULTS Our approach yields limits of detection of acetaldehyde in the nanomolar range and limits of quantification in the low micromolar range. Our linear calibration includes 5 concentrations with a least square regression greater than 0.99 for both acetaldehyde and ethanol. Tissue analyses using this method revealed the capacity to quantify ethanol and acetaldehyde in blood, brain, and liver tissue from mice. CONCLUSIONS By allowing quantification of very low concentrations, this method may be used to examine the formation of ethanol metabolites, specifically acetaldehyde, in murine brain tissue in alcohol research. PMID:27501276

Event-specific real-time detection and quantification of genetically modified Roundup Ready soybean.

PubMed

Huang, Chia-Chia; Pan, Tzu-Ming

2005-05-18

The event-specific real-time detection and quantification of Roundup Ready soybean (RRS) using an ABI PRISM 7700 sequence detection system with light upon extension (LUX) primer was developed in this study. The event-specific primers were designed, targeting the junction of the RRS 5' integration site and the endogenous gene lectin1. Then, a standard reference plasmid was constructed that carried both of the targeted sequences for quantitative analysis. The detection limit of the LUX real-time PCR system was 0.05 ng of 100% RRS genomic DNA, which was equal to 20.5 copies. The range of quantification was from 0.1 to 100%. The sensitivity and range of quantification successfully met the requirement of the labeling rules in the European Union and Taiwan.

Single cell genomic quantification by non-fluorescence nonlinear microscopy

NASA Astrophysics Data System (ADS)

Kota, Divya; Liu, Jing

2017-02-01

Human epidermal growth receptor 2 (Her2) is a gene which plays a major role in breast cancer development. The quantification of Her2 expression in single cells is limited by several drawbacks in existing fluorescence-based single molecule techniques, such as low signal-to-noise ratio (SNR), strong autofluorescence and background signals from biological components. For rigorous genomic quantification, a robust method of orthogonal detection is highly desirable and we demonstrated it by two non-fluorescent imaging techniques -transient absorption microscopy (TAM) and second harmonic generation (SHG). In TAM, gold nanoparticles (AuNPs) are chosen as an orthogonal probes for detection of single molecules which gives background-free quantifications of single mRNA transcript. In SHG, emission from barium titanium oxide (BTO) nanoprobes was demonstrated which allows stable signal beyond the autofluorescence window. Her2 mRNA was specifically labeled with nanoprobes which are conjugated with antibodies or oligonucleotides and quantified at single copy sensitivity in the cancer cells and tissues. Furthermore, a non-fluorescent super-resolution concept, named as second harmonic super-resolution microscopy (SHaSM), was proposed to quantify individual Her2 transcripts in cancer cells beyond the diffraction limit. These non-fluorescent imaging modalities will provide new dimensions in biomarker quantification at single molecule sensitivity in turbid biological samples, offering a strong cross-platform strategy for clinical monitoring at single cell resolution.

Consistency of flow quantifications in tridirectional phase-contrast MRI

NASA Astrophysics Data System (ADS)

Unterhinninghofen, R.; Ley, S.; Dillmann, R.

2009-02-01

Tridirectionally encoded phase-contrast MRI is a technique to non-invasively acquire time-resolved velocity vector fields of blood flow. These may not only be used to analyze pathological flow patterns, but also to quantify flow at arbitrary positions within the acquired volume. In this paper we examine the validity of this approach by analyzing the consistency of related quantifications instead of comparing it with an external reference measurement. Datasets of the thoracic aorta were acquired from 6 pigs, 1 healthy volunteer and 3 patients with artificial aortic valves. Using in-house software an elliptical flow quantification plane was placed manually at 6 positions along the descending aorta where it was rotated to 5 different angles. For each configuration flow was computed based on the original data and data that had been corrected for phase offsets. Results reveal that quantifications are more dependent on changes in position than on changes in angle. Phase offset correction considerably reduces this dependency. Overall consistency is good with a maximum variation coefficient of 9.9% and a mean variation coefficient of 7.2%.

Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

PubMed

Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

2016-02-01

Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Reference gene selection for quantitative real-time PCR in Solanum lycopersicum L. inoculated with the mycorrhizal fungus Rhizophagus irregularis.

PubMed

Fuentes, Alejandra; Ortiz, Javier; Saavedra, Nicolás; Salazar, Luis A; Meneses, Claudio; Arriagada, Cesar

2016-04-01

The gene expression stability of candidate reference genes in the roots and leaves of Solanum lycopersicum inoculated with arbuscular mycorrhizal fungi was investigated. Eight candidate reference genes including elongation factor 1 α (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), protein phosphatase 2A (PP2Acs), ribosomal protein L2 (RPL2), β-tubulin (TUB), ubiquitin (UBI) and actin (ACT) were selected, and their expression stability was assessed to determine the most stable internal reference for quantitative PCR normalization in S. lycopersicum inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. The stability of each gene was analysed in leaves and roots together and separated using the geNorm and NormFinder algorithms. Differences were detected between leaves and roots, varying among the best-ranked genes depending on the algorithm used and the tissue analysed. PGK, TUB and EF1 genes showed higher stability in roots, while EF1 and UBI had higher stability in leaves. Statistical algorithms indicated that the GAPDH gene was the least stable under the experimental conditions assayed. Then, we analysed the expression levels of the LePT4 gene, a phosphate transporter whose expression is induced by fungal colonization in host plant roots. No differences were observed when the most stable genes were used as reference genes. However, when GAPDH was used as the reference gene, we observed an overestimation of LePT4 expression. In summary, our results revealed that candidate reference genes present variable stability in S. lycopersicum arbuscular mycorrhizal symbiosis depending on the algorithm and tissue analysed. Thus, reference gene selection is an important issue for obtaining reliable results in gene expression quantification. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Quantification of regional cerebral blood flow and volume with dynamic susceptibility contrast-enhanced MR imaging.

PubMed

Rempp, K A; Brix, G; Wenz, F; Becker, C R; Gückel, F; Lorenz, W J

1994-12-01

Quantification of regional cerebral blood flow (rCBF) and volume (rCBV) with dynamic magnetic resonance (MR) imaging. After bolus administration of a paramagnetic contrast medium, rapid T2*-weighted gradient-echo images of two sections were acquired for the simultaneous creation of concentration-time curves in the brain-feeding arteries and in brain tissue. Absolute rCBF and rCBV values were determined for gray and white brain matter in 12 subjects with use of principles of the indicator dilution theory. The mean rCBF value in gray matter was 69.7 mL/min +/- 29.7 per 100 g tissue and in white matter, 33.6 mL/min +/- 11.5 per 100 g tissue; the average rCBV was 8.0 mL +/- 3.1 per 100 g tissue and 4.2 mL +/- 1.0 per 100 g tissue, respectively. An age-related decrease in rCBF and rCBV for gray and white matter was observed. Preliminary data demonstrate that the proposed technique allows the quantification of rCBF and rCBV. Although the results are in good agreement with data from positron emission tomography studies, further evaluation is needed to establish the validity of method.

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

PubMed

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

2006-01-01

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

Quantification of Age-Related Lung Tissue Mechanics under Mechanical Ventilation.

PubMed

Kim, JongWon; Heise, Rebecca L; Reynolds, Angela M; Pidaparti, Ramana M

2017-09-29

Elderly patients with obstructive lung diseases often receive mechanical ventilation to support their breathing and restore respiratory function. However, mechanical ventilation is known to increase the severity of ventilator-induced lung injury (VILI) in the elderly. Therefore, it is important to investigate the effects of aging to better understand the lung tissue mechanics to estimate the severity of ventilator-induced lung injuries. Two age-related geometric models involving human bronchioles from generation G10 to G23 and alveolar sacs were developed. The first is for a 50-year-old (normal) and second is for an 80-year old (aged) model. Lung tissue mechanics of normal and aged models were investigated under mechanical ventilation through computational simulations. Results obtained indicated that lung tissue strains during inhalation (t = 0.2 s) decreased by about 40% in the alveolar sac (G23) and 27% in the bronchiole (G20), respectively, for the 80-year-old as compared to the 50-year-old. The respiratory mechanics parameters (work of breathing per unit volume and maximum tissue strain) over G20 and G23 for the 80-year-old decreased by about 64% (three-fold) and 80% (four-fold), respectively, during the mechanical ventilation breathing cycle. However, there was a significant increase (by about threefold) in lung compliance for the 80-year-old in comparison to the 50-year-old. These findings from the computational simulations demonstrated that lung mechanical characteristics are significantly compromised in aging tissues, and these effects were quantified in this study.

Use of diagnostic accuracy as a metric for evaluating laboratory proficiency with microarray assays using mixed-tissue RNA reference samples.

PubMed

Pine, P S; Boedigheimer, M; Rosenzweig, B A; Turpaz, Y; He, Y D; Delenstarr, G; Ganter, B; Jarnagin, K; Jones, W D; Reid, L H; Thompson, K L

2008-11-01

Effective use of microarray technology in clinical and regulatory settings is contingent on the adoption of standard methods for assessing performance. The MicroArray Quality Control project evaluated the repeatability and comparability of microarray data on the major commercial platforms and laid the groundwork for the application of microarray technology to regulatory assessments. However, methods for assessing performance that are commonly applied to diagnostic assays used in laboratory medicine remain to be developed for microarray assays. A reference system for microarray performance evaluation and process improvement was developed that includes reference samples, metrics and reference datasets. The reference material is composed of two mixes of four different rat tissue RNAs that allow defined target ratios to be assayed using a set of tissue-selective analytes that are distributed along the dynamic range of measurement. The diagnostic accuracy of detected changes in expression ratios, measured as the area under the curve from receiver operating characteristic plots, provides a single commutable value for comparing assay specificity and sensitivity. The utility of this system for assessing overall performance was evaluated for relevant applications like multi-laboratory proficiency testing programs and single-laboratory process drift monitoring. The diagnostic accuracy of detection of a 1.5-fold change in signal level was found to be a sensitive metric for comparing overall performance. This test approaches the technical limit for reliable discrimination of differences between two samples using this technology. We describe a reference system that provides a mechanism for internal and external assessment of laboratory proficiency with microarray technology and is translatable to performance assessments on other whole-genome expression arrays used for basic and clinical research.

Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice.

PubMed

Deroost, Katrien; Lays, Natacha; Noppen, Sam; Martens, Erik; Opdenakker, Ghislain; Van den Steen, Philippe E

2012-05-14

Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intra-erythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivo immunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence. An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6 J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann-Whitney U test and Pearsons correlation analysis. Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas sub-nanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels. The developed techniques were useful to quantify Hz in

Detection, mapping, and quantification of single walled carbon nanotubes in histological specimens with photoacoustic microscopy.

PubMed

Avti, Pramod K; Hu, Song; Favazza, Christopher; Mikos, Antonios G; Jansen, John A; Shroyer, Kenneth R; Wang, Lihong V; Sitharaman, Balaji

2012-01-01

In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Optical-resolution (OR) and acoustic-resolution (AR)--Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs.

Quantification of human body fat tissue percentage by MRI.

PubMed

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

2011-01-01

The MRI-based evaluation of the quantity and regional distribution of adipose tissue is one objective measure in the investigation of obesity. The aim of this article was to report a comprehensive and automatic analytical method for the determination of the volumes of subcutaneous fat tissue (SFT) and visceral fat tissue (VFT) in either the whole human body or selected slices or regions of interest. Using an MRI protocol in an examination position that was convenient for volunteers and patients with severe diseases, 22 healthy subjects were examined. The software platform was able to merge MRI scans of several body regions acquired in separate acquisitions. Through a cascade of image processing steps, SFT and VFT volumes were calculated. Whole-body SFT and VFT distributions, as well as fat distributions of defined body slices, were analysed in detail. Complete three-dimensional datasets were analysed in a reproducible manner with as few operator-dependent interventions as possible. In order to determine the SFT volume, the ARTIS (Adapted Rendering for Tissue Intensity Segmentation) algorithm was introduced. The advantage of the ARTIS algorithm was the delineation of SFT volumes in regions in which standard region grow techniques fail. Using the ARTIS algorithm, an automatic SFT volume detection was feasible. MRI data analysis was able to determine SFT and VFT volume percentages using new analytical strategies. With the techniques described, it was possible to detect changes in SFT and VFT percentages of the whole body and selected regions. The techniques presented in this study are likely to be of use in obesity-related investigations, as well as in the examination of longitudinal changes in weight during various medical conditions. Copyright © 2010 John Wiley & Sons, Ltd.

Microfluidic Device to Quantify the Behavior of Therapeutic Bacteria in Three-Dimensional Tumor Tissue.

PubMed

Brackett, Emily L; Swofford, Charles A; Forbes, Neil S

2016-01-01

Microfluidic devices enable precise quantification of the interactions between anti-cancer bacteria and tumor tissue. Direct observation of bacterial movement and gene expression in tissue is difficult with either monolayers of cells or tumor-bearing mice. Quantification of these interactions is necessary to understand the inherent mechanisms of bacterial targeting and to develop modified organisms with enhanced therapeutic properties. Here we describe the procedures for designing, printing, and assembling microfluidic tumor-on-a-chip devices. We also describe the procedures for inserting three-dimensional tumor-cell masses, exposure to bacteria, and analyzing the resultant images.

Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System

NASA Astrophysics Data System (ADS)

Jayasena, T.; Poljak, A.; Braidy, N.; Zhong, L.; Rowlands, B.; Muenchhoff, J.; Grant, R.; Smythe, G.; Teo, C.; Raftery, M.; Sachdev, P.

2016-10-01

Sirtuin proteins have a variety of intracellular targets, thereby regulating multiple biological pathways including neurodegeneration. However, relatively little is currently known about the role or expression of the 7 mammalian sirtuins in the central nervous system. Western blotting, PCR and ELISA are the main techniques currently used to measure sirtuin levels. To achieve sufficient sensitivity and selectivity in a multiplex-format, a targeted mass spectrometric assay was developed and validated for the quantification of all seven mammalian sirtuins (SIRT1-7). Quantification of all peptides was by multiple reaction monitoring (MRM) using three mass transitions per protein-specific peptide, two specific peptides for each sirtuin and a stable isotope labelled internal standard. The assay was applied to a variety of samples including cultured brain cells, mammalian brain tissue, CSF and plasma. All sirtuin peptides were detected in the human brain, with SIRT2 being the most abundant. Sirtuins were also detected in human CSF and plasma, and guinea pig and mouse tissues. In conclusion, we have successfully applied MRM mass spectrometry for the detection and quantification of sirtuin proteins in the central nervous system, paving the way for more quantitative and functional studies.

Noninvasive bi-graphical analysis for the quantification of slowly reversible radioligand binding

NASA Astrophysics Data System (ADS)

Seo, Seongho; Kim, Su Jin; Yoo, Hye Bin; Lee, Jee-Young; Kyeong Kim, Yu; Lee, Dong Soo; Zhou, Yun; Lee, Jae Sung

2016-09-01

In this paper, we presented a novel reference-region-based (noninvasive) bi-graphical analysis for the quantification of a reversible radiotracer binding that may be too slow to reach relative equilibrium (RE) state during positron emission tomography (PET) scans. The proposed method indirectly implements the noninvasive Logan plot, through arithmetic combination of the parameters of two other noninvasive methods and the apparent tissue-to-plasma efflux rate constant for the reference region (k2\\prime ). We investigated its validity and statistical properties, by performing a simulation study with various noise levels and k2\\prime values, and also evaluated its feasibility for [18F]FP-CIT PET in human brain. The results revealed that the proposed approach provides distribution volume ratio estimation comparable to the Logan plot at low noise levels while improving underestimation caused by non-RE state differently depending on k2\\prime . Furthermore, the proposed method was able to avoid noise-induced bias of the Logan plot, and the variability of its results was less dependent on k2\\prime than the Logan plot. Therefore, this approach, without issues related to arterial blood sampling given a pre-estimate of k2\\prime (e.g. population-based), could be useful in parametric image generation for slow kinetic tracers staying in a non-RE state within a PET scan.

A multicenter study benchmarks software tools for label-free proteome quantification.

PubMed

Navarro, Pedro; Kuharev, Jörg; Gillet, Ludovic C; Bernhardt, Oliver M; MacLean, Brendan; Röst, Hannes L; Tate, Stephen A; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I; Aebersold, Ruedi; Tenzer, Stefan

2016-11-01

Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.

Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis.

PubMed

Gilhodes, Jean-Claude; Julé, Yvon; Kreuz, Sebastian; Stierstorfer, Birgit; Stiller, Detlef; Wollin, Lutz

2017-01-01

Current literature on pulmonary fibrosis induced in animal models highlights the need of an accurate, reliable and reproducible histological quantitative analysis. One of the major limits of histological scoring concerns the fact that it is observer-dependent and consequently subject to variability, which may preclude comparative studies between different laboratories. To achieve a reliable and observer-independent quantification of lung fibrosis we developed an automated software histological image analysis performed from digital image of entire lung sections. This automated analysis was compared to standard evaluation methods with regard to its validation as an end-point measure of fibrosis. Lung fibrosis was induced in mice by intratracheal administration of bleomycin (BLM) at 0.25, 0.5, 0.75 and 1 mg/kg. A detailed characterization of BLM-induced fibrosis was performed 14 days after BLM administration using lung function testing, micro-computed tomography and Ashcroft scoring analysis. Quantification of fibrosis by automated analysis was assessed based on pulmonary tissue density measured from thousands of micro-tiles processed from digital images of entire lung sections. Prior to analysis, large bronchi and vessels were manually excluded from the original images. Measurement of fibrosis has been expressed by two indexes: the mean pulmonary tissue density and the high pulmonary tissue density frequency. We showed that tissue density indexes gave access to a very accurate and reliable quantification of morphological changes induced by BLM even for the lowest concentration used (0.25 mg/kg). A reconstructed 2D-image of the entire lung section at high resolution (3.6 μm/pixel) has been performed from tissue density values allowing the visualization of their distribution throughout fibrotic and non-fibrotic regions. A significant correlation (p<0.0001) was found between automated analysis and the above standard evaluation methods. This correlation establishes

Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis

PubMed Central

Gilhodes, Jean-Claude; Kreuz, Sebastian; Stierstorfer, Birgit; Stiller, Detlef; Wollin, Lutz

2017-01-01

Current literature on pulmonary fibrosis induced in animal models highlights the need of an accurate, reliable and reproducible histological quantitative analysis. One of the major limits of histological scoring concerns the fact that it is observer-dependent and consequently subject to variability, which may preclude comparative studies between different laboratories. To achieve a reliable and observer-independent quantification of lung fibrosis we developed an automated software histological image analysis performed from digital image of entire lung sections. This automated analysis was compared to standard evaluation methods with regard to its validation as an end-point measure of fibrosis. Lung fibrosis was induced in mice by intratracheal administration of bleomycin (BLM) at 0.25, 0.5, 0.75 and 1 mg/kg. A detailed characterization of BLM-induced fibrosis was performed 14 days after BLM administration using lung function testing, micro-computed tomography and Ashcroft scoring analysis. Quantification of fibrosis by automated analysis was assessed based on pulmonary tissue density measured from thousands of micro-tiles processed from digital images of entire lung sections. Prior to analysis, large bronchi and vessels were manually excluded from the original images. Measurement of fibrosis has been expressed by two indexes: the mean pulmonary tissue density and the high pulmonary tissue density frequency. We showed that tissue density indexes gave access to a very accurate and reliable quantification of morphological changes induced by BLM even for the lowest concentration used (0.25 mg/kg). A reconstructed 2D-image of the entire lung section at high resolution (3.6 μm/pixel) has been performed from tissue density values allowing the visualization of their distribution throughout fibrotic and non-fibrotic regions. A significant correlation (p<0.0001) was found between automated analysis and the above standard evaluation methods. This correlation establishes

Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

PubMed

Floren, C; Wiedemann, I; Brenig, B; Schütz, E; Beck, J

2015-04-15

Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications.

PubMed

Kolacsek, Orsolya; Pergel, Enikő; Varga, Nóra; Apáti, Ágota; Orbán, Tamás I

2017-01-20

There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the ΔΔCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of tissue viability imaging and colorimetry: skin blanching.

PubMed

Zhai, Hongbo; Chan, Heidi P; Farahmand, Sara; Nilsson, Gert E; Maibach, Howard I

2009-02-01

Operator-independent assessment of skin blanching is important in the development and evaluation of topically applied steroids. Spectroscopic instruments based on hand-held probes, however, include elements of operator dependence such as difference in applied pressure and probe misalignment, while laser Doppler-based methods are better suited for demonstration of skin vasodilatation than for vasoconstriction. To demonstrate the potential of the emerging technology of Tissue Viability Imaging (TiVi) in the objective and operator-independent assessment of skin blanching. The WheelsBridge TiVi600 Tissue Viability Imager was used for quantification of human skin blanching with the Minolta chromameter CR 200 as an independent colorimeter reference method. Desoximetasone gel 0.05% was applied topically on the volar side of the forearm under occlusion for 6 h in four healthy adults. In a separate study, the induction of blanching in the occlusion phase was mapped using a transparent occlusion cover. The relative uncertainty in the blanching estimate produced by the Tissue Viability Imager was about 5% and similar to that of the chromameter operated by a single user and taking the a(*) parameter as a measure of blanching. Estimation of skin blanching could also be performed in the presence of a transient paradoxical erythema, using the integrated TiVi software. The successive induction of skin blanching during the occlusion phase could readily be mapped by the Tissue Viability Imager. TiVi seems to be suitable for operator-independent and remote mapping of human skin blanching, eliminating the main disadvantages of methods based on hand-held probes.

Evaluation of New Reference Genes in Papaya for Accurate Transcript Normalization under Different Experimental Conditions

PubMed Central

Chen, Weixin; Chen, Jianye; Lu, Wangjin; Chen, Lei; Fu, Danwen

2012-01-01

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

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

PubMed

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

2014-12-01

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

Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification.

PubMed

Ghedira, Rim; Papazova, Nina; Vuylsteke, Marnik; Ruttink, Tom; Taverniers, Isabel; De Loose, Marc

2009-10-28

GMO quantification, based on real-time PCR, relies on the amplification of an event-specific transgene assay and a species-specific reference assay. The uniformity of the nucleotide sequences targeted by both assays across various transgenic varieties is an important prerequisite for correct quantification. Single nucleotide polymorphisms (SNPs) frequently occur in the maize genome and might lead to nucleotide variation in regions used to design primers and probes for reference assays. Further, they may affect the annealing of the primer to the template and reduce the efficiency of DNA amplification. We assessed the effect of a minor DNA template modification, such as a single base pair mismatch in the primer attachment site, on real-time PCR quantification. A model system was used based on the introduction of artificial mismatches between the forward primer and the DNA template in the reference assay targeting the maize starch synthase (SSIIb) gene. The results show that the presence of a mismatch between the primer and the DNA template causes partial to complete failure of the amplification of the initial DNA template depending on the type and location of the nucleotide mismatch. With this study, we show that the presence of a primer/template mismatch affects the estimated total DNA quantity to a varying degree.

HPLC Quantification of astaxanthin and canthaxanthin in Salmonidae eggs.

PubMed

Tzanova, Milena; Argirova, Mariana; Atanasov, Vasil

2017-04-01

Astaxanthin and canthaxanthin are naturally occurring antioxidants referred to as xanthophylls. They are used as food additives in fish farms to improve the organoleptic qualities of salmonid products and to prevent reproductive diseases. This study reports the development and single-laboratory validation of a rapid method for quantification of astaxanthin and canthaxanthin in eggs of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis М.). An advantage of the proposed method is the perfect combination of selective extraction of the xanthophylls and analysis of the extract by high-performance liquid chromatography and photodiode array detection. The method validation was carried out in terms of linearity, accuracy, precision, recovery and limits of detection and quantification. The method was applied for simultaneous quantification of the two xanthophylls in eggs of rainbow trout and brook trout after their selective extraction. The results show that astaxanthin accumulations in salmonid fish eggs are larger than those of canthaxanthin. As the levels of these two xanthophylls affect fish fertility, this method can be used to improve the nutritional quality and to minimize the occurrence of the M74 syndrome in fish populations. Copyright © 2016 John Wiley & Sons, Ltd.

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

PubMed

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

2015-10-10

A UPLC-MS method was developed for determination of pterostilbene (PTS) in plasma and tissues of mice. PTS was separated on Agilent Zorbax XDB-C18 column (50 × 2.1 mm, 1.8 μm) with gradient mobile phase at the flow rate of 0.2 ml/min. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode. The linear calibration curve of PTS in mouse plasma and tissues ranged from 1.0 to 5000 and 0.50 to 500 ng/ml (r(2)>0.9979), respectively, with lowest limits of quantification (LLOQ) were between 0.5 and 2.0 ng/ml, respectively. The accuracy and precision of the assay were satisfactory. The validated method was applied to the study of bioavailability and tissue distribution of PTS in normal and Lewis lung carcinoma (LLC) bearing mice. The bioavailability of PTS (dose 14, 28 and 56 mg/kg) in normal mice were 11.9%, 13.9% and 26.4%, respectively; and the maximum level (82.1 ± 14.2 μg/g) was found in stomach (dose 28 mg/kg). The bioavailability, peak concentration (Cmax), time to peak concentration (Tmax) of PTS in LLC mice was increased compared with normal mice. The results indicated the UPLC-MS method is reliable and bioavailability and tissue distribution of PTS in normal and LLC mice were dramatically different. Copyright © 2015 Elsevier B.V. All rights reserved.

Uncertainty Quantification for Robust Control of Wind Turbines using Sliding Mode Observer

NASA Astrophysics Data System (ADS)

Schulte, Horst

2016-09-01

A new quantification method of uncertain models for robust wind turbine control using sliding-mode techniques is presented with the objective to improve active load mitigation. This approach is based on the so-called equivalent output injection signal, which corresponds to the average behavior of the discontinuous switching term, establishing and maintaining a motion on a so-called sliding surface. The injection signal is directly evaluated to obtain estimates of the uncertainty bounds of external disturbances and parameter uncertainties. The applicability of the proposed method is illustrated by the quantification of a four degree-of-freedom model of the NREL 5MW reference turbine containing uncertainties.

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

PubMed

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

2016-08-01

For 3-dimensional (3D) imaging of a tissue, 3 methodological steps are essential and their successful application depends on specific characteristics of the type of tissue. The steps are 1° clearing of the opaque tissue to render it transparent for microscopy, 2° fluorescence labeling of the tissues and 3° 3D imaging. In the past decades, new methodologies were introduced for the clearing steps with their specific advantages and disadvantages. Most clearing techniques have been applied to the central nervous system and other organs that contain relatively low amounts of connective tissue including extracellular matrix. However, tissues that contain large amounts of extracellular matrix such as dermis in skin or gingiva are difficult to clear. The present survey lists methodologies that are available for clearing of tissues for 3D imaging. We report here that the BABB method using a mixture of benzyl alcohol and benzyl benzoate and iDISCO using dibenzylether (DBE) are the most successful methods for clearing connective tissue-rich gingiva and dermis of skin for 3D histochemistry and imaging of fluorescence using light-sheet microscopy. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Quantification of lithium at ppm level in geological samples using nuclear reaction analysis.

PubMed

De La Rosa, Nathaly; Kristiansson, Per; Nilsson, E J Charlotta; Ros, Linus; Pallon, Jan; Skogby, Henrik

2018-01-01

Proton-induced reaction (p,α) is one type of nuclear reaction analysis (NRA) suitable especially for light element quantification. In the case of lithium quantification presented in this work, accelerated protons with an energy about of 850 keV were used to induce the 7 Li(p,α) 4 He reaction in standard reference and geological samples such as tourmaline and other Li-minerals. It is shown that this technique for lithium quantification allowed for measurement of concentrations down below one ppm. The possibility to relate the lithium content with the boron content in a single analysis was also demonstrated using tourmaline samples, both in absolute concentration and in lateral distribution. In addition, Particle induced X-ray emission (PIXE) was utilized as a complementary IBA technique for simultaneous mapping of elements heavier than sodium.

A rapid method for identification and quantification of prostaglandins in cerebral tissues by UHPLC-ESI-MS/MS for the lipidomic in vivo studies.

PubMed

Gobo, Luciana Assis; de Carvalho, Leandro Machado; Temp, Fernanda; Viana, Carine; Mello, Carlos Fernando

2018-03-15

An analytical method utilizing liquid chromatography coupled to mass spectrometry with electrospray ionization has been developed for the identification of prostaglandins (PGs) in cerebral tissues. The five compounds identified (thromboxane B2, prostaglandin E2, prostaglandin D2, 6-keto-prostaglandin F1 alpha and prostaglandin F2 alpha) are cellular mediators of inflammation and are involved in a variety of physiological and pathological processes by acting on membrane receptors on the surfaces of target cells. The parameters of the electrospray ionization interface were optimized to obtain the highest possible sensitivity for all compounds studied. The limits of detection ranged from 0.25 to 1.09 μg L -1 , and the limits of quantification ranged from 0.83 to 3.64 μg L -1 . The method was validated and applied to samples of brain tissue from five mice. The sample concentrations of the four prostaglandins quantified ranged from 375 ȵg L -1 for prostaglandin E2 to 6602 μg L -1 for prostaglandin D2. An advantage of this work that should be emphasized is the fast response of the method, which allows to obtaining the lipid profile after a 3 min chromatographic run. Copyright © 2018 Elsevier Inc. All rights reserved.

Detection, Mapping, and Quantification of Single Walled Carbon Nanotubes in Histological Specimens with Photoacoustic Microscopy

PubMed Central

Mikos, Antonios G.; Jansen, John A.; Shroyer, Kenneth R.; Wang, Lihong V.; Sitharaman, Balaji

2012-01-01

Aims In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Materials and Methods Optical-resolution (OR) and acoustic-resolution (AR) - Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Results Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. Conclusions The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs. PMID:22496892

Evaluation of reference gene suitability for quantitative expression analysis by quantitative polymerase chain reaction in the mandibular condyle of sheep.

PubMed

Jiang, Xin; Xue, Yang; Zhou, Hongzhi; Li, Shouhong; Zhang, Zongmin; Hou, Rui; Ding, Yuxiang; Hu, Kaijin

2015-10-01

Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression. Suitable reference genes belonging to numerous functional classes have been identified for various types of species and tissue. However, little is currently known regarding the most suitable reference genes for bone, specifically for the sheep mandibular condyle. Sheep are important for the study of human bone diseases, particularly for temporomandibular diseases. The present study aimed to identify a set of reference genes suitable for the normalization of qPCR data from the mandibular condyle of sheep. A total of 12 reference genes belonging to various functional classes were selected, and the expression stability of the reference genes was determined in both the normal and fractured area of the sheep mandibular condyle. RefFinder, which integrates the following currently available computational algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, was used to compare and rank the candidate reference genes. The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle. As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle. In addition, ACTB and PGK1 may be considered suitable alternatives.

One Novel Multiple-Target Plasmid Reference Molecule Targeting Eight Genetically Modified Canola Events for Genetically Modified Canola Detection.

PubMed

Li, Zhuqing; Li, Xiang; Wang, Canhua; Song, Guiwen; Pi, Liqun; Zheng, Lan; Zhang, Dabing; Yang, Litao

2017-09-27

Multiple-target plasmid DNA reference materials have been generated and utilized as good substitutes of matrix-based reference materials in the analysis of genetically modified organisms (GMOs). Herein, we report the construction of one multiple-target plasmid reference molecule, pCAN, which harbors eight GM canola event-specific sequences (RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45) and a partial sequence of the canola endogenous reference gene PEP. The applicability of this plasmid reference material in qualitative and quantitative PCR assays of the eight GM canola events was evaluated, including the analysis of specificity, limit of detection (LOD), limit of quantification (LOQ), and performance of pCAN in the analysis of various canola samples, etc. The LODs are 15 copies for RF2, MS1, and RT73 assays using pCAN as the calibrator and 10 genome copies for the other events. The LOQ in each event-specific real-time PCR assay is 20 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and PEP assays are between 91% and 97%, and the squared regression coefficients (R 2 ) are all higher than 0.99. The quantification bias values varied from 0.47% to 20.68% with relative standard deviation (RSD) from 1.06% to 24.61% in the quantification of simulated samples. Furthermore, 10 practical canola samples sampled from imported shipments in the port of Shanghai, China, were analyzed employing pCAN as the calibrator, and the results were comparable with those assays using commercial certified materials as the calibrator. Concluding from these results, we believe that this newly developed pCAN plasmid is one good candidate for being a plasmid DNA reference material in the detection and quantification of the eight GM canola events in routine analysis.

Calibration of BCR-ABL1 mRNA quantification methods using genetic reference materials is a valid strategy to report results on the international scale.

PubMed

Mauté, Carole; Nibourel, Olivier; Réa, Delphine; Coiteux, Valérie; Grardel, Nathalie; Preudhomme, Claude; Cayuela, Jean-Michel

2014-09-01

Until recently, diagnostic laboratories that wanted to report on the international scale had limited options: they had to align their BCR-ABL1 quantification methods through a sample exchange with a reference laboratory to derive a conversion factor. However, commercial methods calibrated on the World Health Organization genetic reference panel are now available. We report results from a study designed to assess the comparability of the two alignment strategies. Sixty follow-up samples from chronic myeloid leukemia patients were included. Two commercial methods calibrated on the genetic reference panel were compared to two conversion factor methods routinely used at Saint-Louis Hospital, Paris, and at Lille University Hospital. Results were matched against concordance criteria (i.e., obtaining at least two of the three following landmarks: 50, 75 and 90% of the patient samples within a 2-fold, 3-fold and 5-fold range, respectively). Out of the 60 samples, more than 32 were available for comparison. Compared to the conversion factor method, the two commercial methods were within a 2-fold, 3-fold and 5-fold range for 53 and 59%, 89 and 88%, 100 and 97%, respectively of the samples analyzed at Saint-Louis. At Lille, results were 45 and 85%, 76 and 97%, 100 and 100%, respectively. Agreements between methods were observed in the four comparisons performed. Our data show that the two commercial methods selected are concordant with the conversion factor methods. This study brings the proof of principle that alignment on the international scale using the genetic reference panel is compatible with the patient sample exchange procedure. We believe that these results are particularly important for diagnostic laboratories wishing to adopt commercial methods. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2009-04-01

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

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

PubMed Central

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

2009-01-01

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

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

PubMed

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

2014-10-10

The red abalone Haliotis rufescens is one of the most important species for aquaculture in Baja California, México, and despite this, few gene expression studies have been done in tissues such as gill, head and gonad. For this purpose, reverse transcription and quantitative real time PCR (RT-qPCR) is a powerful tool for gene expression evaluation. For a reliable analysis, however, it is necessary to select and validate housekeeping genes that allow proper transcription quantification. Stability of nine housekeeping genes (ACTB, BGLU, TUBB, CY, GAPDH, HPRTI, RPL5, SDHA and UBC) was evaluated in different tissues of red abalone (gill, head and gonad/digestive gland). Four-fold serial dilutions of cDNA (from 25 ngμL(-1) to 0.39 ngμL(-1)) were used to prepare the standard curve, and it showed gene efficiencies between 0.95 and 0.99, with R(2)=0.99. geNorm and NormFinder analysis showed that RPL5 and CY were the most stable genes considering all tissues, whereas in gill HPRTI and BGLU were most stable. In gonad/digestive gland, RPL5 and TUBB were the most stable genes with geNorm, while SDHA and HPRTI were the best using NormFinder. Similarly, in head the best genes were RPL5 and UBC with geNorm, and GAPDH and CY with NormFinder. The technical variability analysis with RPL5 and abalone gonad/digestive gland tissue indicated a high repeatability with a variation coefficient within groups ≤ 0.56% and between groups ≤ 1.89%. These results will help us for further research in reproduction, thermoregulation and endocrinology in red abalone. Copyright © 2014 Elsevier B.V. All rights reserved.

Reference Standardization for Mass Spectrometry and High-resolution Metabolomics Applications to Exposome Research

PubMed Central

Go, Young-Mi; Walker, Douglas I.; Liang, Yongliang; Uppal, Karan; Soltow, Quinlyn A.; Tran, ViLinh; Strobel, Frederick; Quyyumi, Arshed A.; Ziegler, Thomas R.; Pennell, Kurt D.; Miller, Gary W.; Jones, Dean P.

2015-01-01

The exposome is the cumulative measure of environmental influences and associated biological responses throughout the lifespan, including exposures from the environment, diet, behavior, and endogenous processes. A major challenge for exposome research lies in the development of robust and affordable analytic procedures to measure the broad range of exposures and associated biologic impacts occurring over a lifetime. Biomonitoring is an established approach to evaluate internal body burden of environmental exposures, but use of biomonitoring for exposome research is often limited by the high costs associated with quantification of individual chemicals. High-resolution metabolomics (HRM) uses ultra-high resolution mass spectrometry with minimal sample preparation to support high-throughput relative quantification of thousands of environmental, dietary, and microbial chemicals. HRM also measures metabolites in most endogenous metabolic pathways, thereby providing simultaneous measurement of biologic responses to environmental exposures. The present research examined quantification strategies to enhance the usefulness of HRM data for cumulative exposome research. The results provide a simple reference standardization protocol in which individual chemical concentrations in unknown samples are estimated by comparison to a concurrently analyzed, pooled reference sample with known chemical concentrations. The approach was tested using blinded analyses of amino acids in human samples and was found to be comparable to independent laboratory results based on surrogate standardization or internal standardization. Quantification was reproducible over a 13-month period and extrapolated to thousands of chemicals. The results show that reference standardization protocol provides an effective strategy that will enhance data collection for cumulative exposome research. In principle, the approach can be extended to other types of mass spectrometry and other analytical methods. PMID

Direct Quantification of Solute Diffusivity in Agarose and Articular Cartilage Using Correlation Spectroscopy.

PubMed

Shoga, Janty S; Graham, Brian T; Wang, Liyun; Price, Christopher

2017-10-01

Articular cartilage is an avascular tissue; diffusive transport is critical for its homeostasis. While numerous techniques have been used to quantify diffusivity within porous, hydrated tissues and tissue engineered constructs, these techniques have suffered from issues regarding invasiveness and spatial resolution. In the present study, we implemented and compared two separate correlation spectroscopy techniques, fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), for the direct, and minimally-invasive quantification of fluorescent solute diffusion in agarose and articular cartilage. Specifically, we quantified the diffusional properties of fluorescein and Alexa Fluor 488-conjugated dextrans (3k and 10k) in aqueous solutions, agarose gels of varying concentration (i.e. 1, 3, 5%), and in different zones of juvenile bovine articular cartilage explants (i.e. superficial, middle, and deep). In agarose, properties of solute diffusion obtained via FCS and RICS were inversely related to molecule size, gel concentration, and applied strain. In cartilage, the diffusional properties of solutes were similarly dependent upon solute size, cartilage zone, and compressive strain; findings that agree with work utilizing other quantification techniques. In conclusion, this study established the utility of FCS and RICS as simple and minimally invasive techniques for quantifying microscale solute diffusivity within agarose constructs and articular cartilage explants.

Diagnostic value of virtual touch tissue imaging quantification for benign and malignant breast lesions with different sizes

PubMed Central

Liu, Hui; Zhao, Li-Xia; Xu, Guang; Yao, Ming-Hua; Zhang, Ai-Hong; Xu, Hui-Xiong; Wu, Rong

2015-01-01

The study was to explore diagnostic value of the virtual touch tissue imaging quantification (VTIQ) in distinguishing benign and malignant breast lesions of variable sizes. We performed conventional ultrasound and VTIQ in 139 breast lesions. The lesions were categorized into three groups according to size (group 1, ≤ 10 mm; group 2, 10-20 mm; and group 3, > 20 mm), and their mean, min, and max shear wave velocities (SWVs) were measured. Diagnoses were confirmed by pathological examination after surgery or needle biopsy. Receiver-operating characteristic curves (ROC) were constructed to determine the optimum cut-off values, calculate the area under curve (AUC), the sensitivity, specificity and accuracy for each velocity. For all groups, the mean, min, and max SWVs of malignant lesions were significantly higher than those of benign lesions (P < 0.05). The cut-off values of mean, min, and max SWVs were not significantly different among the three groups. In addition, the diagnostic performance of mean, min, and max SWV values is analogous, regardless of lesion size. In conclusion, VTIQ is a strong complement to conventional ultrasound, which is a promising method in the differential diagnosis of the breast lesions with different sizes. Further studies validate our results as well as reduce the number of unnecessary biopsies, regardless of size is warranted. PMID:26550234

Validation of an LC-MS/MS method for the quantification of choline-related compounds and phospholipids in foods and tissues.

PubMed

Xiong, Yeping; Zhao, Yuan-Yuan; Goruk, Sue; Oilund, Kirsten; Field, Catherine J; Jacobs, René L; Curtis, Jonathan M

2012-12-12

A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) method was developed and validated to simultaneously quantify six aqueous choline-related compounds and eight major phospholipids classes in a single run. HILIC chromatography was coupled to positive ion electrospray mass spectrometry. A combination of multiple scan modes including precursor ion scan, neutral loss scan and multiple reaction monitoring was optimized for the determination of each compound or class in a single LC/MS run. This work developed a simplified extraction scheme in which both free choline and related compounds along with phospholipids were extracted into a homogenized phase using chloroform/methanol/water (1:2:0.8) and diluted into methanol for the analysis of target compounds in a variety of sample matrices. The analyte recoveries were evaluated by spiking tissues and food samples with two isotope-labeled internal standards, PC-d(3) and Cho-d(3). Recoveries of between 90% and 115% were obtained by spiking a range of sample matrices with authentic standards containing all 14 of the target analytes. The precision of the analysis ranged from 1.6% to 13%. Accuracy and precision was comparable to that obtained by quantification of selected phospholipid classes using (31)P NMR. A variety of sample matrices including egg yolks, human diets and animal tissues were analyzed using the validated method. The measurements of total choline in selected foods were found to be in good agreement with values obtained from the USDA choline database. Copyright © 2012 Elsevier B.V. All rights reserved.

Reference Materials: Significance, General Requirements, and Demand.

PubMed

Kiełbasa, Anna; Gadzała-Kopciuch, Renata; Buszewski, Bogusław

2016-05-03

Reference materials play an important part in the quality control of measurements. Rapid development of such new scientific disciplines as proteomics, metabolomics, and genomics also necessitates development of new reference materials. This is a great challenge due to the complexity of the production of new reference materials and difficulties associated with achieving their homogeneity and stability. CRMs of tissue are of particular importance. They can be counted among the matrices that are most complex and time consuming in preparation. Tissue is the place of transformation and accumulation of many substances (e.g., metabolites, which are intermediate or end products resulting from metabolic processes). Trace amounts of many substances in tissues must be determined with adequate precision and accuracy. To meet the needs stemming from research and from problems and challenges faced by chemists, analysts, and toxicologists, the number of certified reference materials should be continuously increased.

Methods for quantifying adipose tissue insulin resistance in overweight/obese humans.

PubMed

Ter Horst, K W; van Galen, K A; Gilijamse, P W; Hartstra, A V; de Groot, P F; van der Valk, F M; Ackermans, M T; Nieuwdorp, M; Romijn, J A; Serlie, M J

2017-08-01

Insulin resistance of adipose tissue is an important feature of obesity-related metabolic disease. However, assessment of lipolysis in humans requires labor-intensive and expensive methods, and there is limited validation of simplified measurement methods. We aimed to validate simplified methods for the quantification of adipose tissue insulin resistance against the assessment of insulin sensitivity of lipolysis suppression during hyperinsulinemic-euglycemic clamp studies. We assessed the insulin-mediated suppression of lipolysis by tracer-dilution of [1,1,2,3,3- 2 H 5 ]glycerol during hyperinsulinemic-euglycemic clamp studies in 125 overweight or obese adults (85 men, 40 women; age 50±11 years; body mass index 38±7 kg m -2 ). Seven indices of adipose tissue insulin resistance were validated against the reference measurement method. Low-dose insulin infusion resulted in suppression of the glycerol rate of appearance ranging from 4% (most resistant) to 85% (most sensitive), indicating a good range of adipose tissue insulin sensitivity in the study population. The reference method correlated with (1) insulin-mediated suppression of plasma glycerol concentrations (r=0.960, P<0.001), (2) suppression of plasma non-esterified fatty acid (NEFA) concentrations (r=0.899, P<0.001), (3) the Adipose tissue Insulin Resistance (Adipo-IR) index (fasting plasma insulin-NEFA product; r=-0.526, P<0.001), (4) the fasting plasma insulin-glycerol product (r=-0.467, P<0.001), (5) the Adipose Tissue Insulin Resistance Index (fasting plasma insulin-basal lipolysis product; r=0.460, P<0.001), (6) the Quantitative Insulin Sensitivity Check Index (QUICKI)-NEFA index (r=0.621, P<0.001), and (7) the QUICKI-glycerol index (r=0.671, P<0.001). Bland-Altman plots showed no systematic errors for the suppression indices but proportional errors for all fasting indices. Receiver-operator characteristic curves confirmed that all indices were able to detect adipose tissue insulin resistance (area

The Impact of Repeated Freeze-Thaw Cycles on the Quality of Biomolecules in Four Different Tissues.

PubMed

Ji, Xiaoli; Wang, Min; Li, Lingling; Chen, Fang; Zhang, Yanyang; Li, Qian; Zhou, Junmei

2017-10-01

High-quality biosamples are valuable resources for biomedical research. However, some tissues are stored without being sectioned into small aliquots and have to undergo repeated freeze-thaw cycles throughout prolonged experimentation. Little is known regarding the effects of repeated freeze-thaw cycles on the quality of biomolecules in tissues. The aim of this study was to evaluate the impact of repeated freeze-thaw (at room temperature or on ice) cycles on biomolecules and gene expression in four different types of tissues. Each fresh tissue was sectioned into seven aliquots and snap-frozen before undergoing repeated freeze-thaw cycles at room temperature or on ice. Biomolecules were extracted and analyzed. Both relative and absolute quantification were used to detect the changes in gene expression. The results indicated that the impact of repeated freeze-thaw cycles on RNA integrity varied by tissue type. Gene expression, including the housekeeping gene, was affected in RNA-degraded samples according to absolute quantification rather than relative quantification. Furthermore, our results suggest that thawing on ice could protect RNA integrity compared with thawing at room temperature. No obvious degradation of protein or DNA was observed with repeated freeze-thaw cycles either at room temperature or on ice. This research provides ample evidence for the necessity of sectioning fresh tissues into small aliquots before snap-freezing, thus avoiding degradation of RNA and alteration of gene expression resulting from repeated freeze-thaw cycles. For frozen tissue samples that were already in storage and had to be used repeatedly during their lifecycle, thawing on ice or sectioned at ultralow temperature is recommended.

Sensitivity of Chemical Shift-Encoded Fat Quantification to Calibration of Fat MR Spectrum

PubMed Central

Wang, Xiaoke; Hernando, Diego; Reeder, Scott B.

2015-01-01

Purpose To evaluate the impact of different fat spectral models on proton density fat-fraction (PDFF) quantification using chemical shift-encoded (CSE) MRI. Material and Methods Simulations and in vivo imaging were performed. In a simulation study, spectral models of fat were compared pairwise. Comparison of magnitude fitting and mixed fitting was performed over a range of echo times and fat fractions. In vivo acquisitions from 41 patients were reconstructed using 7 published spectral models of fat. T2-corrected STEAM-MRS was used as reference. Results Simulations demonstrate that imperfectly calibrated spectral models of fat result in biases that depend on echo times and fat fraction. Mixed fitting is more robust against this bias than magnitude fitting. Multi-peak spectral models showed much smaller differences among themselves than when compared to the single-peak spectral model. In vivo studies show all multi-peak models agree better (for mixed fitting, slope ranged from 0.967–1.045 using linear regression) with reference standard than the single-peak model (for mixed fitting, slope=0.76). Conclusion It is essential to use a multi-peak fat model for accurate quantification of fat with CSE-MRI. Further, fat quantification techniques using multi-peak fat models are comparable and no specific choice of spectral model is shown to be superior to the rest. PMID:25845713

Quantification of mixed chimerism by real time PCR on whole blood-impregnated FTA cards.

PubMed

Pezzoli, N; Silvy, M; Woronko, A; Le Treut, T; Lévy-Mozziconacci, A; Reviron, D; Gabert, J; Picard, C

2007-09-01

This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.

Dynamic impact indentation of hydrated biological tissues and tissue surrogate gels

NASA Astrophysics Data System (ADS)

Ilke Kalcioglu, Z.; Qu, Meng; Strawhecker, Kenneth E.; Shazly, Tarek; Edelman, Elazer; VanLandingham, Mark R.; Smith, James F.; Van Vliet, Krystyn J.

2011-03-01

For both materials engineering research and applied biomedicine, a growing need exists to quantify mechanical behaviour of tissues under defined hydration and loading conditions. In particular, characterisation under dynamic contact-loading conditions can enable quantitative predictions of deformation due to high rate 'impact' events typical of industrial accidents and ballistic insults. The impact indentation responses were examined of both hydrated tissues and candidate tissue surrogate materials. The goals of this work were to determine the mechanical response of fully hydrated soft tissues under defined dynamic loading conditions, and to identify design principles by which synthetic, air-stable polymers could mimic those responses. Soft tissues from two organs (liver and heart), a commercially available tissue surrogate gel (Perma-Gel™) and three styrenic block copolymer gels were investigated. Impact indentation enabled quantification of resistance to penetration and energy dissipative constants under the rates and energy densities of interest for tissue surrogate applications. These analyses indicated that the energy dissipation capacity under dynamic impact increased with increasing diblock concentration in the styrenic gels. Under the impact rates employed (2 mm/s to 20 mm/s, corresponding to approximate strain energy densities from 0.4 kJ/m3 to 20 kJ/m3), the energy dissipation capacities of fully hydrated soft tissues were ultimately well matched by a 50/50 triblock/diblock composition that is stable in ambient environments. More generally, the methodologies detailed here facilitate further optimisation of impact energy dissipation capacity of polymer-based tissue surrogate materials, either in air or in fluids.

HCV-RNA quantification in liver bioptic samples and extrahepatic compartments, using the abbott RealTime HCV assay.

PubMed

Antonucci, FrancescoPaolo; Cento, Valeria; Sorbo, Maria Chiara; Manuelli, Matteo Ciancio; Lenci, Ilaria; Sforza, Daniele; Di Carlo, Domenico; Milana, Martina; Manzia, Tommaso Maria; Angelico, Mario; Tisone, Giuseppe; Perno, Carlo Federico; Ceccherini-Silberstein, Francesca

2017-08-01

We evaluated the performance of a rapid method to quantify HCV-RNA in the hepatic and extrahepatic compartments, by using for the first time the Abbott RealTime HCV-assay. Non-tumoral (NT), tumoral (TT) liver samples, lymph nodes and ascitic fluid from patients undergoing orthotopic-liver-transplantation (N=18) or liver resection (N=4) were used for the HCV-RNA quantification; 5/22 patients were tested after or during direct acting antivirals (DAA) treatment. Total RNA and DNA quantification from tissue-biopsies allowed normalization of HCV-RNA concentrations in IU/μg of total RNA and IU/10 6 liver-cells, respectively. HCV-RNA was successfully quantified with high reliability in liver biopsies, lymph nodes and ascitic fluid samples. Among the 17 untreated patients, a positive and significant HCV-RNA correlation between serum and NT liver-samples was observed (Pearson: rho=0.544, p=0.024). Three DAA-treated patients were HCV-RNA "undetectable" in serum, but still "detectable" in all tested liver-tissues. Differently, only one DAA-treated patient, tested after sustained-virological-response, showed HCV-RNA "undetectability" in liver-tissue. HCV-RNA was successfully quantified with high reliability in liver bioptic samples and extrahepatic compartments, even when HCV-RNA was "undetectable" in serum. Abbott RealTime HCV-assay is a good diagnostic tool for HCV quantification in intra- and extra-hepatic compartments, whenever a bioptic sample is available. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

brain 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 [(18)F]-florbetaben PET. Hindbrain white matter reference performed best when considering the composite of quality criteria.

Target analyte quantification by isotope dilution LC-MS/MS directly referring to internal standard concentrations--validation for serum cortisol measurement.

PubMed

Maier, Barbara; Vogeser, Michael

2013-04-01

Isotope dilution LC-MS/MS methods used in the clinical laboratory typically involve multi-point external calibration in each analytical series. Our aim was to test the hypothesis that determination of target analyte concentrations directly derived from the relation of the target analyte peak area to the peak area of a corresponding stable isotope labelled internal standard compound [direct isotope dilution analysis (DIDA)] may be not inferior to conventional external calibration with respect to accuracy and reproducibility. Quality control samples and human serum pools were analysed in a comparative validation protocol for cortisol as an exemplary analyte by LC-MS/MS. Accuracy and reproducibility were compared between quantification either involving a six-point external calibration function, or a result calculation merely based on peak area ratios of unlabelled and labelled analyte. Both quantification approaches resulted in similar accuracy and reproducibility. For specified analytes, reliable analyte quantification directly derived from the ratio of peak areas of labelled and unlabelled analyte without the need for a time consuming multi-point calibration series is possible. This DIDA approach is of considerable practical importance for the application of LC-MS/MS in the clinical laboratory where short turnaround times often have high priority.

A study of a tissue equivalent gelatine based tissue substitute

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

Spence, J.L.

1992-11-01

A study of several tissue substitutes for use as volumetric dosimeters was performed. The tissue substitutes studied included tissue substitutes from previous studies and from ICRU 44. The substitutes were evaluated for an overall match to Reference Man which was used as a basis for this study. The evaluation was based on the electron stopping power, the mass attenuation coefficient, the electron density, and the specific gravity. The tissue substitute chosen also had to be capable of changing from a liquid into a solid form to maintain an even distribution of thermoluminesent dosimetry (TLD) powder and then back to amore » liquid for recovery of the TLD powder without adversely effecting the TLD powder. The gelatine mixture provided the closest match to the data from Reference Man tissue. The gelatine mixture was put through a series of test to determine it's usefulness as a reliable tissue substitute. The TLD powder was cast in the gelatine mixture and recovered to determine if the TLD powder was adversely effected. The distribution of the TLD powder after being cast into the gelatin mixture was tested in insure an even was maintained.« less

A study of a tissue equivalent gelatine based tissue substitute

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

Spence, Jody L.

1992-11-01

A study of several tissue substitutes for use as volumetric dosimeters was performed. The tissue substitutes studied included tissue substitutes from previous studies and from ICRU 44. The substitutes were evaluated for an overall match to Reference Man which was used as a basis for this study. The evaluation was based on the electron stopping power, the mass attenuation coefficient, the electron density, and the specific gravity. The tissue substitute chosen also had to be capable of changing from a liquid into a solid form to maintain an even distribution of thermoluminesent dosimetry (TLD) powder and then back to amore » liquid for recovery of the TLD powder without adversely effecting the TLD powder. The gelatine mixture provided the closest match to the data from Reference Man tissue. The gelatine mixture was put through a series of test to determine it`s usefulness as a reliable tissue substitute. The TLD powder was cast in the gelatine mixture and recovered to determine if the TLD powder was adversely effected. The distribution of the TLD powder after being cast into the gelatin mixture was tested in insure an even was maintained.« less

Sulfur-based absolute quantification of proteins using isotope dilution inductively coupled plasma mass spectrometry

NASA Astrophysics Data System (ADS)

Lee, Hyun-Seok; Heun Kim, Sook; Jeong, Ji-Seon; Lee, Yong-Moon; Yim, Yong-Hyeon

2015-10-01

An element-based reductive approach provides an effective means of realizing International System of Units (SI) traceability for high-purity biological standards. Here, we develop an absolute protein quantification method using double isotope dilution (ID) inductively coupled plasma mass spectrometry (ICP-MS) combined with microwave-assisted acid digestion for the first time. We validated the method and applied it to certify the candidate protein certified reference material (CRM) of human growth hormone (hGH). The concentration of hGH was determined by analysing the total amount of sulfur in hGH. Next, the size-exclusion chromatography method was used with ICP-MS to characterize and quantify sulfur-containing impurities. By subtracting the contribution of sulfur-containing impurities from the total sulfur content in the hGH CRM, we obtained a SI-traceable certification value. The quantification result obtained with the present method based on sulfur analysis was in excellent agreement with the result determined via a well-established protein quantification method based on amino acid analysis using conventional acid hydrolysis combined with an ID liquid chromatography-tandem mass spectrometry. The element-based protein quantification method developed here can be generally used for SI-traceable absolute quantification of proteins, especially pure-protein standards.

Reference Standardization for Mass Spectrometry and High-resolution Metabolomics Applications to Exposome Research.

PubMed

Go, Young-Mi; Walker, Douglas I; Liang, Yongliang; Uppal, Karan; Soltow, Quinlyn A; Tran, ViLinh; Strobel, Frederick; Quyyumi, Arshed A; Ziegler, Thomas R; Pennell, Kurt D; Miller, Gary W; Jones, Dean P

2015-12-01

The exposome is the cumulative measure of environmental influences and associated biological responses throughout the lifespan, including exposures from the environment, diet, behavior, and endogenous processes. A major challenge for exposome research lies in the development of robust and affordable analytic procedures to measure the broad range of exposures and associated biologic impacts occurring over a lifetime. Biomonitoring is an established approach to evaluate internal body burden of environmental exposures, but use of biomonitoring for exposome research is often limited by the high costs associated with quantification of individual chemicals. High-resolution metabolomics (HRM) uses ultra-high resolution mass spectrometry with minimal sample preparation to support high-throughput relative quantification of thousands of environmental, dietary, and microbial chemicals. HRM also measures metabolites in most endogenous metabolic pathways, thereby providing simultaneous measurement of biologic responses to environmental exposures. The present research examined quantification strategies to enhance the usefulness of HRM data for cumulative exposome research. The results provide a simple reference standardization protocol in which individual chemical concentrations in unknown samples are estimated by comparison to a concurrently analyzed, pooled reference sample with known chemical concentrations. The approach was tested using blinded analyses of amino acids in human samples and was found to be comparable to independent laboratory results based on surrogate standardization or internal standardization. Quantification was reproducible over a 13-month period and extrapolated to thousands of chemicals. The results show that reference standardization protocol provides an effective strategy that will enhance data collection for cumulative exposome research. In principle, the approach can be extended to other types of mass spectrometry and other analytical methods. © The

Reference ranges for regional cerebral tissue oxygen saturation and fractional oxygen extraction in neonates during immediate transition after birth.

PubMed

Pichler, Gerhard; Binder, Corinna; Avian, Alexander; Beckenbach, Elisabeth; Schmölzer, Georg M; Urlesberger, Berndt

2013-12-01

To define reference ranges for regional cerebral tissue oxygen saturation (crSO2) and regional cerebral fractional tissue oxygen extraction (cFTOE) during the first 15 minutes after birth in neonates requiring no medical support. The crSO2 was measured using near infrared spectroscopy (Invos 5100 cerebral/somatic oximeter monitor; Somanetics Corp, Troy, Michigan) during the first 15 minutes after birth for term and preterm neonates. The near infrared spectroscopy sensor was placed on the left forehead. Peripheral oxygen saturation and heart rate were continuously measured by pulse oximetry, and cFTOE was calculated. Neonates were excluded if they required any medical support. A total of 381 neonates were included: 82 term neonates after vaginal delivery, 272 term neonates after cesarean delivery, and 27 preterm neonates after cesarean delivery. In all neonates, median (10th-90th percentiles) crSO2 was 41% (23-64) at 2 minutes, 68% (45-85) at 5 minutes, 79% (65-90) at 10 minutes, and 77% (63-89) at 15 minutes of age. In all neonates, median (10th-90th percentiles) cFTOE was 33% (11-70) at 2 minutes, 21% (6-45) at 5 minutes, 15% (5-31) at 10 minutes, and 18% (7-34) at 15 minutes of age. We report reference ranges of crSO2 and cFTOE in neonates requiring no medical support during transition immediately after birth. The use of cerebral oxygenation monitoring and use of these reference ranges in neonates during transition may help to guide oxygen delivery and avoid cerebral hypo-oxygenation and hyperoxygenation. Copyright © 2013 Mosby, Inc. All rights reserved.

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).

Large scale systematic proteomic quantification from non-metastatic to metastatic colorectal cancer

NASA Astrophysics Data System (ADS)

Yin, Xuefei; Zhang, Yang; Guo, Shaowen; Jin, Hong; Wang, Wenhai; Yang, Pengyuan

2015-07-01

A systematic proteomic quantification of formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues from stage I to stage IIIC was performed in large scale. 1017 proteins were identified with 338 proteins in quantitative changes by label free method, while 341 proteins were quantified with significant expression changes among 6294 proteins by iTRAQ method. We found that proteins related to migration expression increased and those for binding and adherent decreased during the colorectal cancer development according to the gene ontology (GO) annotation and ingenuity pathway analysis (IPA). The integrin alpha 5 (ITA5) in integrin family was focused, which was consistent with the metastasis related pathway. The expression level of ITA5 decreased in metastasis tissues and the result has been further verified by Western blotting. Another two cell migration related proteins vitronectin (VTN) and actin-related protein (ARP3) were also proved to be up-regulated by both mass spectrometry (MS) based quantification results and Western blotting. Up to now, our result shows one of the largest dataset in colorectal cancer proteomics research. Our strategy reveals a disease driven omics-pattern for the metastasis colorectal cancer.

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. © 2014 John Wiley & Sons Ltd.

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers.

PubMed

Dong, Lianhua; Meng, Ying; Wang, Jing; Liu, Yingying

2014-02-01

DNA reference materials of certified value have a critical function in many analytical processes of DNA measurement. Quantification of amoA genes in ammonia oxidizing bacteria (AOB) and archaea (AOA), and of nirS and nosZ genes in the denitrifiers is very important for determining their distribution and abundance in the natural environment. A plasmid reference material containing nirS, nosZ, amoA-AOB, and amoA-AOA is developed to provide a DNA standard with copy number concentration for ensuring comparability and reliability of quantification of these genes. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material. The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR. Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR. Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was used to provide independent data for quantifying the plasmid reference material. The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC-IDMS, improving both the accuracy and reliability of the plasmid reference material. The reference value, with its expanded uncertainty (k = 2), of the plasmid reference material was determined to be (5.19 ± 0.41) × 10(9) copies μL(-1) by averaging the results of two independent measurements. Consideration of the factors revealed in this study can improve the reliability and accuracy of ddPCR; thus, this method has the potential to accurately quantify DNA reference materials.

Evaluation and Selection of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Withania somnifera (L.) Dunal

PubMed Central

Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar

2015-01-01

Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

Radio-frequency energy quantification in magnetic resonance imaging

NASA Astrophysics Data System (ADS)

Alon, Leeor

Mapping of radio frequency (RF) energy deposition has been challenging for 50+ years, especially, when scanning patients in the magnetic resonance imaging (MRI) environment. As result, electromagnetic simulation software is often used for estimating the specific absorption rate (SAR), the rate of RF energy deposition in tissue. The thesis work presents challenges associated with aligning information provided by electromagnetic simulation and MRI experiments. As result of the limitations of simulations, experimental methods for the quantification of SAR were established. A system for quantification of the total RF energy deposition was developed for parallel transmit MRI (a system that uses multiple antennas to excite and image the body). The system is capable of monitoring and predicting channel-by-channel RF energy deposition, whole body SAR and capable of tracking potential hardware failures that occur in the transmit chain and may cause the deposition of excessive energy into patients. Similarly, we demonstrated that local RF power deposition can be mapped and predicted for parallel transmit systems based on a series of MRI temperature mapping acquisitions. Resulting from the work, we developed tools for optimal reconstruction temperature maps from MRI acquisitions. The tools developed for temperature mapping paved the way for utilizing MRI as a diagnostic tool for evaluation of RF/microwave emitting device safety. Quantification of the RF energy was demonstrated for both MRI compatible and non-MRI-compatible devices (such as cell phones), while having the advantage of being noninvasive, of providing millimeter resolution and high accuracy.

Nanoscale X-Ray Microscopic Imaging of Mammalian Mineralized Tissue

PubMed Central

Andrews, Joy C.; Almeida, Eduardo; van der Meulen, Marjolein C.H.; Alwood, Joshua S.; Lee, Chialing; Liu, Yijin; Chen, Jie; Meirer, Florian; Feser, Michael; Gelb, Jeff; Rudati, Juana; Tkachuk, Andrei; Yun, Wenbing; Pianetta, Piero

2010-01-01

A novel hard transmission X-ray microscope (TXM) at the Stanford Synchrotron Radiation Light-source operating from 5 to 15 keV X-ray energy with 14 to 30 µm2 field of view has been used for high-resolution (30–40 nm) imaging and density quantification of mineralized tissue. TXM is uniquely suited for imaging of internal cellular structures and networks in mammalian mineralized tissues using relatively thick (50 µm), untreated samples that preserve tissue micro- and nanostructure. To test this method we performed Zernike phase contrast and absorption contrast imaging of mouse cancellous bone prepared under different conditions of in vivo loading, fixation, and contrast agents. In addition, the three-dimensional structure was examined using tomography. Individual osteocytic lacunae were observed embedded within trabeculae in cancellous bone. Extensive canalicular networks were evident and included processes with diameters near the 30–40 nm instrument resolution that have not been reported previously. Trabecular density was quantified relative to rod-like crystalline apatite, and rod-like trabecular struts were found to have 51–54% of pure crystal density and plate-like areas had 44–53% of crystal density. The nanometer resolution of TXM enables future studies for visualization and quantification of ultrastructural changes in bone tissue resulting from osteoporosis, dental disease, and other pathologies. PMID:20374681

In vivo quantification of spatially-varying mechanical properties in developing tissues

PubMed Central

Serwane, Friedhelm; Mongera, Alessandro; Rowghanian, Payam; Kealhofer, David A.; Lucio, Adam A.; Hockenbery, Zachary M.; Campàs, Otger

2017-01-01

It is generally believed that the mechanical properties of the cellular microenvironment and their spatiotemporal variations play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has been performed yet. Here we introduce a technique that employs biocompatible ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo. Using this technique, we show that vertebrate body elongation entails spatially-varying tissue mechanics along the anteroposterior axis. Specifically, we find that the zebrafish tailbud is viscoelastic (elastic below a few seconds and fluid after just one minute) and displays decreasing stiffness and increasing fluidity towards its posterior elongating region. This method opens new avenues to study mechanobiology in vivo, both in embryogenesis and in disease processes, including cancer. PMID:27918540

Nondestructive quantification of analyte diffusion in cornea and sclera using optical coherence tomography.

PubMed

Ghosn, Mohamad G; Tuchin, Valery V; Larin, Kirill V

2007-06-01

Noninvasive functional imaging, monitoring, and quantification of analytes transport in epithelial ocular tissues are extremely important for therapy and diagnostics of many eye diseases. In this study the authors investigated the capability of optical coherence tomography (OCT) for noninvasive monitoring and quantification of diffusion of different analytes in sclera and cornea of rabbit eyes. A portable time-domain OCT system with wavelength of 1310 +/- 15 nm, output power of 3.5 mW, and resolution of 25 mum was used in this study. Diffusion of different analytes was monitored and quantified in rabbit cornea and sclera of whole eyeballs. Diffusion of water, metronidazole (0.5%), dexamethasone (0.2%), ciprofloxacin (0.3%), mannitol (20%), and glucose solution (20%) were examined, and their permeability coefficients were calculated by using OCT signal slope and depth-resolved amplitude methods. Permeability coefficients were calculated as a function of time and tissue depth. For instance, mannitol was found to have a permeability coefficient of (8.99 +/- 1.43) x 10(-6) cm/s in cornea and (6.18 +/- 1.08) x 10(-6) cm/s in sclera. The permeability coefficient of drugs with small concentrations (where water was the major solvent) was found to be in the range of that of water in the same tissue type, whereas permeability coefficients of higher concentrated solutions varied significantly. Results suggest that the OCT technique might be a powerful tool for noninvasive diffusion studies of different analytes in ocular tissues. However, additional methods of OCT signal acquisition and processing are required to study the diffusion of agents of small concentrations.

Quantification and localization of mast cells in periapical lesions.

PubMed

Mahita, V N; Manjunatha, B S; Shah, R; Astekar, M; Purohit, S; Kovvuru, S

2015-01-01

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

Quantification and Localization of Mast Cells in Periapical Lesions

PubMed Central

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

2015-01-01

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

Tylosin depletion from edible pig tissues.

PubMed

Prats, C; El Korchi, G; Francesch, R; Arboix, M; Pérez, B

2002-12-01

The depletion of tylosin from edible pig tissues was studied following 5 days of intramuscular (i.m.) administration of 10 mg/kg of tylosin to 16 crossbreed pigs. Animals were slaughtered at intervals after treatment and samples of muscle, kidney, liver, skin+fat, and injection site were collected and analysed by high-performance liquid chromatography (HPLC). Seven days after the completion of treatment, the concentration of tylosin in kidney, skin+fat, and at the injection site was higher than the European Union maximal residue limit (MRL) of 100 microg/kg. Tylosin residues in all tissues were below the quantification limit (50 microg/kg) at 10 and 14 days post-treatment.

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.

TH-CD-202-05: DECT Based Tissue Segmentation as Input to Monte Carlo Simulations for Proton Treatment Verification Using PET Imaging

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

Berndt, B; Wuerl, M; Dedes, G

Purpose: To improve agreement of predicted and measured positron emitter yields in patients, after proton irradiation for PET-based treatment verification, using a novel dual energy CT (DECT) tissue segmentation approach, overcoming known deficiencies from single energy CT (SECT). Methods: DECT head scans of 5 trauma patients were segmented and compared to existing decomposition methods with a first focus on the brain. For validation purposes, three brain equivalent solutions [water, white matter (WM) and grey matter (GM) – equivalent with respect to their reference carbon and oxygen contents and CT numbers at 90kVp and 150kVp] were prepared from water, ethanol, sucrosemore » and salt. The activities of all brain solutions, measured during a PET scan after uniform proton irradiation, were compared to Monte Carlo simulations. Simulation inputs were various solution compositions obtained from different segmentation approaches from DECT, SECT scans, and known reference composition. Virtual GM solution salt concentration corrections were applied based on DECT measurements of solutions with varying salt concentration. Results: The novel tissue segmentation showed qualitative improvements in %C for patient brain scans (ground truth unavailable). The activity simulations based on reference solution compositions agree with the measurement within 3–5% (4–8Bq/ml). These reference simulations showed an absolute activity difference between WM (20%C) and GM (10%C) to H2O (0%C) of 43 Bq/ml and 22 Bq/ml, respectively. Activity differences between reference simulations and segmented ones varied from −6 to 1 Bq/ml for DECT and −79 to 8 Bq/ml for SECT. Conclusion: Compared to the conventionally used SECT segmentation, the DECT based segmentation indicates a qualitative and quantitative improvement. In controlled solutions, a MC input based on DECT segmentation leads to better agreement with the reference. Future work will address the anticipated improvement of

A multi-center study benchmarks software tools for label-free proteome quantification

PubMed Central

Gillet, Ludovic C; Bernhardt, Oliver M.; MacLean, Brendan; Röst, Hannes L.; Tate, Stephen A.; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I.; Aebersold, Ruedi; Tenzer, Stefan

2016-01-01

The consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra), a method that uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test datasets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation windows setups. For consistent evaluation we developed LFQbench, an R-package to calculate metrics of precision and accuracy in label-free quantitative MS, and report the identification performance, robustness and specificity of each software tool. Our reference datasets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics. PMID:27701404

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.

Fast and sensitive HPLC/UV method for cefazolin quantification in plasma and subcutaneous tissue microdialysate of humans and rodents applied to pharmacokinetic studies in obese individuals.

PubMed

Palma, Eduardo Celia; Laureano, João Victor; de Araújo, Bibiana Verlindo; Meinhardt, Nelson Guardiola; Stein, Airton Tetelbom; Dalla Costa, Teresa

2018-04-14

Antimicrobial prophylactic dosing of morbidly obese patients may differ from normal weighted individuals owing to alterations in drug tissue distribution. Drug subcutaneous tissue distribution can be investigated by microdialysis patients and animals. The need for cefazolin prophylactic dose adjustment in obese patients remains under discussion. The paper describes the validation of an HPLC-UV method for cefazolin quantification in plasma and microdialysate samples from clinical and pre-clinical studies. A C 18 column with an isocratic mobile phase was used for drug separation, with detection at 272 nm. Total and unbound cefazolin lower limit of quantitation was 5 μg/mL in human plasma, 2 μg/mL in rat plasma, and 0.5 and 0.025 μg/mL in human and rat microdialysate samples, respectively. The maximum intra- and inter-day imprecisions were 10.7 and 8.1%, respectively. The inaccuracy was <9.7%. The limit of quantitation imprecision and inaccuracy were < 15%. Cefazolin stability in the experimental conditions was confirmed. Cefazolin plasma concentrations and subcutaneous tissue penetration were determined by microdialysis in morbidly obese patients (2 g i.v. bolus) and diet-induced obese rats (30 mg/kg i.v. bolus) using the method. This method has the main advantages of easy plasma clean-up and practicability and has proven to be useful in cefazolin clinical and pre-clinical pharmacokinetic investigations. Copyright © 2018 John Wiley & Sons, Ltd.

Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L.

PubMed

Moreira, Viviane S; Soares, Virgínia L F; Silva, Raner J S; Sousa, Aurizangela O; Otoni, Wagner C; Costa, Marcio G C

2018-05-01

Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B. orellana , coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that RPL38 is the most stable gene in different tissues and stages of seed development and 18SrRNA is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable RPL38 and the least stable gene, 18SrRNA , for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the CCD1 gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.

Quantification of cardiolipin by liquid chromatography-electrospray ionization mass spectrometry.

PubMed

Garrett, Teresa A; Kordestani, Reza; Raetz, Christian R H

2007-01-01

Cardiolipin (CL), a tetra-acylated glycerophospholipid composed of two phosphatidyl moieties linked by a bridging glycerol, plays an important role in mitochondrial function in eukaryotic cells. Alterations to the content and acylation state of CL cause mitochondrial dysfunction and may be associated with pathologies such as ischemia, hypothyrodism, aging, and heart failure. The structure of CL is very complex because of microheterogeneity among its four acyl chains. Here we have developed a method for the quantification of CL molecular species by liquid chromatography-electrospray ionization mass spectrometry. We quantify the [M-2H](2-) ion of a CL of a given molecular formula and identify the CLs by their total number of carbons and unsaturations in the acyl chains. This method, developed using mouse macrophage RAW 264.7 tumor cells, is broadly applicable to other cell lines, tissues, bacteria and yeast. Furthermore, this method could be used for the quantification of lyso-CLs and bis-lyso-CLs.

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

Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes.

PubMed

Salvi, Sergio; D'Orso, Fabio; Morelli, Giorgio

2008-06-25

Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.

Managing tissue bank access to the OR for tissue recovery.

PubMed

Trim, Robert S

2011-08-01

Hospitals often have tissue recovery service agreements with regional tissue banks to facilitate the donation process. The agreements that outline the tissue bank-hospital relationship frequently allow tissue bank personnel to perform tissue recovery procedures in the referring hospital's OR and may or may not specify any preparatory orientation or any written protocols for tissue bank staff members to follow. This creates the potential for unintentional breaches of protocol that can affect operation of equipment or result in contamination that may put surgical patients and staff members at risk. The OR manager is responsible for establishing appropriate orientation plans for tissue bank employees to ensure they understand and adhere to the hospital's protocols. Copyright © 2011 AORN, Inc. Published by Elsevier Inc. All rights reserved.

Analysis of street drugs in seized material without primary reference standards.

PubMed

Laks, Suvi; Pelander, Anna; Vuori, Erkki; Ali-Tolppa, Elisa; Sippola, Erkki; Ojanperä, Ilkka

2004-12-15

A novel approach was used to analyze street drugs in seized material without primary reference standards. Identification was performed by liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS), essentially based on accurate mass determination using a target library of 735 exact monoisotopic masses. Quantification was carried out by liquid chromatography/chemiluminescence nitrogen detection (LC/CLND) with a single secondary standard (caffeine), utilizing the detector's equimolar response to nitrogen. Sample preparation comprised dilution, first with methanol and further with the LC mobile phase. Altogether 21 seized drug samples were analyzed blind by the present method, and results were compared to accredited reference methods utilizing identification by gas chromatography/mass spectrometry and quantification by gas chromatography or liquid chromatography. The 31 drug findings by LC/TOFMS comprised 19 different drugs-of-abuse, byproducts, and adulterants, including amphetamine and tryptamine designer drugs, with one unresolved pair of compounds having an identical mass. By the reference methods, 27 findings could be confirmed, and among the four unconfirmed findings, only 1 apparent false positive was found. In the quantitative analysis of 11 amphetamine, heroin, and cocaine findings, mean relative difference between the results of LC/CLND and the reference methods was 11% (range 4.2-21%), without any observable bias. Mean relative standard deviation for three parallel LC/CLND results was 6%. Results suggest that the present combination of LC/TOFMS and LC/CLND offers a simple solution for the analysis of scheduled and designer drugs in seized material, independent of the availability of primary reference standards.

Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

PubMed

Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

2016-01-01

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

Zooming In on Plant Hormone Analysis: Tissue- and Cell-Specific Approaches.

PubMed

Novák, Ondřej; Napier, Richard; Ljung, Karin

2017-04-28

Plant hormones are a group of naturally occurring, low-abundance organic compounds that influence physiological processes in plants. Our knowledge of the distribution profiles of phytohormones in plant organs, tissues, and cells is still incomplete, but advances in mass spectrometry have enabled significant progress in tissue- and cell-type-specific analyses of phytohormones over the last decade. Mass spectrometry is able to simultaneously identify and quantify hormones and their related substances. Biosensors, on the other hand, offer continuous monitoring; can visualize local distributions and real-time quantification; and, in the case of genetically encoded biosensors, are noninvasive. Thus, biosensors offer additional, complementary technologies for determining temporal and spatial changes in phytohormone concentrations. In this review, we focus on recent advances in mass spectrometry-based quantification, describe monitoring systems based on biosensors, and discuss validations of the various methods before looking ahead at future developments for both approaches.

Breast density quantification using magnetic resonance imaging (MRI) with bias field correction: A postmortem study

PubMed Central

Ding, Huanjun; Johnson, Travis; Lin, Muqing; Le, Huy Q.; Ducote, Justin L.; Su, Min-Ying; Molloi, Sabee

2013-01-01

Purpose: Quantification of breast density based on three-dimensional breast MRI may provide useful information for the early detection of breast cancer. However, the field inhomogeneity can severely challenge the computerized image segmentation process. In this work, the effect of the bias field in breast density quantification has been investigated with a postmortem study. Methods: T1-weighted images of 20 pairs of postmortem breasts were acquired on a 1.5 T breast MRI scanner. Two computer-assisted algorithms were used to quantify the volumetric breast density. First, standard fuzzy c-means (FCM) clustering was used on raw images with the bias field present. Then, the coherent local intensity clustering (CLIC) method estimated and corrected the bias field during the iterative tissue segmentation process. Finally, FCM clustering was performed on the bias-field-corrected images produced by CLIC method. The left–right correlation for breasts in the same pair was studied for both segmentation algorithms to evaluate the precision of the tissue classification. Finally, the breast densities measured with the three methods were compared to the gold standard tissue compositions obtained from chemical analysis. The linear correlation coefficient, Pearson's r, was used to evaluate the two image segmentation algorithms and the effect of bias field. Results: The CLIC method successfully corrected the intensity inhomogeneity induced by the bias field. In left–right comparisons, the CLIC method significantly improved the slope and the correlation coefficient of the linear fitting for the glandular volume estimation. The left–right breast density correlation was also increased from 0.93 to 0.98. When compared with the percent fibroglandular volume (%FGV) from chemical analysis, results after bias field correction from both the CLIC the FCM algorithms showed improved linear correlation. As a result, the Pearson's r increased from 0.86 to 0.92 with the bias field correction

Breast density quantification using magnetic resonance imaging (MRI) with bias field correction: a postmortem study.

PubMed

Ding, Huanjun; Johnson, Travis; Lin, Muqing; Le, Huy Q; Ducote, Justin L; Su, Min-Ying; Molloi, Sabee

2013-12-01

Quantification of breast density based on three-dimensional breast MRI may provide useful information for the early detection of breast cancer. However, the field inhomogeneity can severely challenge the computerized image segmentation process. In this work, the effect of the bias field in breast density quantification has been investigated with a postmortem study. T1-weighted images of 20 pairs of postmortem breasts were acquired on a 1.5 T breast MRI scanner. Two computer-assisted algorithms were used to quantify the volumetric breast density. First, standard fuzzy c-means (FCM) clustering was used on raw images with the bias field present. Then, the coherent local intensity clustering (CLIC) method estimated and corrected the bias field during the iterative tissue segmentation process. Finally, FCM clustering was performed on the bias-field-corrected images produced by CLIC method. The left-right correlation for breasts in the same pair was studied for both segmentation algorithms to evaluate the precision of the tissue classification. Finally, the breast densities measured with the three methods were compared to the gold standard tissue compositions obtained from chemical analysis. The linear correlation coefficient, Pearson's r, was used to evaluate the two image segmentation algorithms and the effect of bias field. The CLIC method successfully corrected the intensity inhomogeneity induced by the bias field. In left-right comparisons, the CLIC method significantly improved the slope and the correlation coefficient of the linear fitting for the glandular volume estimation. The left-right breast density correlation was also increased from 0.93 to 0.98. When compared with the percent fibroglandular volume (%FGV) from chemical analysis, results after bias field correction from both the CLIC the FCM algorithms showed improved linear correlation. As a result, the Pearson's r increased from 0.86 to 0.92 with the bias field correction. The investigated CLIC method

TH-CD-206-01: Expectation-Maximization Algorithm-Based Tissue Mixture Quantification for Perfusion MRI

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

Han, H; Xing, L; Liang, Z

Purpose: To investigate the feasibility of estimating the tissue mixture perfusions and quantifying cerebral blood flow change in arterial spin labeled (ASL) perfusion MR images. Methods: The proposed perfusion MR image analysis framework consists of 5 steps: (1) Inhomogeneity correction was performed on the T1- and T2-weighted images, which are available for each studied perfusion MR dataset. (2) We used the publicly available FSL toolbox to strip off the non-brain structures from the T1- and T2-weighted MR images. (3) We applied a multi-spectral tissue-mixture segmentation algorithm on both T1- and T2-structural MR images to roughly estimate the fraction of eachmore » tissue type - white matter, grey matter and cerebral spinal fluid inside each image voxel. (4) The distributions of the three tissue types or tissue mixture across the structural image array are down-sampled and mapped onto the ASL voxel array via a co-registration operation. (5) The presented 4-dimensional expectation-maximization (4D-EM) algorithm takes the down-sampled three tissue type distributions on perfusion image data to generate the perfusion mean, variance and percentage images for each tissue type of interest. Results: Experimental results on three volunteer datasets demonstrated that the multi-spectral tissue-mixture segmentation algorithm was effective to initialize tissue mixtures from T1- and T2-weighted MR images. Compared with the conventional ASL image processing toolbox, the proposed 4D-EM algorithm not only generated comparable perfusion mean images, but also produced perfusion variance and percentage images, which the ASL toolbox cannot obtain. It is observed that the perfusion contribution percentages may not be the same as the corresponding tissue mixture volume fractions estimated in the structural images. Conclusion: A specific application to brain ASL images showed that the presented perfusion image analysis method is promising for detecting subtle changes in tissue

The principles of quantification applied to in vivo proton MR spectroscopy.

PubMed

Helms, Gunther

2008-08-01

Following the identification of metabolite signals in the in vivo MR spectrum, quantification is the procedure to estimate numerical values of their concentrations. The two essential steps are discussed in detail: analysis by fitting a model of prior knowledge, that is, the decomposition of the spectrum into the signals of singular metabolites; then, normalization of these signals to yield concentration estimates. Special attention is given to using the in vivo water signal as internal reference.

QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification.

PubMed

Simpson, Deborah M; Beynon, Robert J

2012-09-01

Systems biology requires knowledge of the absolute amounts of proteins in order to model biological processes and simulate the effects of changes in specific model parameters. Quantification concatamers (QconCATs) are established as a method to provide multiplexed absolute peptide standards for a set of target proteins in isotope dilution standard experiments. Two or more quantotypic peptides representing each of the target proteins are concatenated into a designer gene that is metabolically labelled with stable isotopes in Escherichia coli or other cellular or cell-free systems. Co-digestion of a known amount of QconCAT with the target proteins generates a set of labelled reference peptide standards for the unlabelled analyte counterparts, and by using an appropriate mass spectrometry platform, comparison of the intensities of the peptide ratios delivers absolute quantification of the encoded peptides and in turn the target proteins for which they are surrogates. In this review, we discuss the criteria and difficulties associated with surrogate peptide selection and provide examples in the design of QconCATs for quantification of the proteins of the nuclear factor κB pathway.

[Muscles and connective tissue: histology].

PubMed

Delage, J-P

2012-10-01

Here, we give some comments about the DVD movies "Muscle Attitudes" from Endovivo productions, the movies up lighting some loss in the attention given to studies on the connective tissue, and especially them into muscles. The main characteristics of the different components in the intra-muscular connective tissue (perimysium, endomysium, epimysium) are shown here with special references to their ordered architecture and special references to their spatial distributions. This connective tissue is abundant into the muscles and is in continuity with the muscles in vicinity, with their tendons and their sheath, sticking the whole on skin. This connective tissue has also very abundant connections on the muscles fibres. It is then assumed that the connective tissue sticks every organs or cells of the locomotion system. Considering the elastic properties of the collagen fibres which are the most abundant component of connective tissue, it is possible to up light a panel of connective tissue associated functions such as the transmission of muscle contractions or the regulation of protein and energetic muscles metabolism. Copyright © 2012. Published by Elsevier SAS.

Multiple products monitoring as a robust approach for peptide quantification.

PubMed

Baek, Je-Hyun; Kim, Hokeun; Shin, Byunghee; Yu, Myeong-Hee

2009-07-01

Quantification of target peptides and proteins is crucial for biomarker discovery. Approaches such as selected reaction monitoring (SRM) and multiple reaction monitoring (MRM) rely on liquid chromatography and mass spectrometric analysis of defined peptide product ions. These methods are not very widespread because the determination of quantifiable product ion using either SRM or MRM is a very time-consuming process. We developed a novel approach for quantifying target peptides without such an arduous process of ion selection. This method is based on monitoring multiple product ions (multiple products monitoring: MpM) from full-range MS2 spectra of a target precursor. The MpM method uses a scoring system that considers both the absolute intensities of product ions and the similarities between the query MS2 spectrum and the reference MS2 spectrum of the target peptide. Compared with conventional approaches, MpM greatly improves sensitivity and selectivity of peptide quantification using an ion-trap mass spectrometer.

Canine body composition quantification using 3 tesla fat-water MRI.

PubMed

Gifford, Aliya; Kullberg, Joel; Berglund, Johan; Malmberg, Filip; Coate, Katie C; Williams, Phillip E; Cherrington, Alan D; Avison, Malcolm J; Welch, E Brian

2014-02-01

To test the hypothesis that a whole-body fat-water MRI (FWMRI) protocol acquired at 3 Tesla combined with semi-automated image analysis techniques enables precise volume and mass quantification of adipose, lean, and bone tissue depots that agree with static scale mass and scale mass changes in the context of a longitudinal study of large-breed dogs placed on an obesogenic high-fat, high-fructose diet. Six healthy adult male dogs were scanned twice, at weeks 0 (baseline) and 4, of the dietary regiment. FWMRI-derived volumes of adipose tissue (total, visceral, and subcutaneous), lean tissue, and cortical bone were quantified using a semi-automated approach. Volumes were converted to masses using published tissue densities. FWMRI-derived total mass corresponds with scale mass with a concordance correlation coefficient of 0.931 (95% confidence interval = [0.813, 0.975]), and slope and intercept values of 1.12 and -2.23 kg, respectively. Visceral, subcutaneous and total adipose tissue masses increased significantly from weeks 0 to 4, while neither cortical bone nor lean tissue masses changed significantly. This is evidenced by a mean percent change of 70.2% for visceral, 67.0% for subcutaneous, and 67.1% for total adipose tissue. FWMRI can precisely quantify and map body composition with respect to adipose, lean, and bone tissue depots. The described approach provides a valuable tool to examine the role of distinct tissue depots in an established animal model of human metabolic disease. Copyright © 2013 Wiley Periodicals, Inc.

Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains.

PubMed

Mehle, Nataša; Dobnik, David; Ravnikar, Maja; Pompe Novak, Maruša

2018-05-03

RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.

Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view.

PubMed

Guehrs, Erik; Schneider, Michael; Günther, Christian M; Hessing, Piet; Heitz, Karen; Wittke, Doreen; López-Serrano Oliver, Ana; Jakubowski, Norbert; Plendl, Johanna; Eisebitt, Stefan; Haase, Andrea

2017-03-21

Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches. We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 μg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in large agglomerates, only a few were found in clusters of fewer than five NPs. Furthermore, we cross-checked our results by using inductively coupled plasma mass spectrometry and could confirm the FIB/SEM results. Our approach based on FIB/SEM slice and view is currently the only one that allows the quantification of the absolute dose of silver NPs in individual cells and at the same time to assess their intracellular distribution at high resolution. We therefore propose to use FIB/SEM slice and view to systematically analyse the cellular uptake of various NPs as a function of size, concentration and incubation time.

Development of magnetic resonance technology for noninvasive boron quantification

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

Bradshaw, K.M.

1990-11-01

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

Tissue polarimetry: concepts, challenges, applications, and outlook.

PubMed

Ghosh, Nirmalya; Vitkin, I Alex

2011-11-01

Polarimetry has a long and successful history in various forms of clear media. Driven by their biomedical potential, the use of the polarimetric approaches for biological tissue assessment has also recently received considerable attention. Specifically, polarization can be used as an effective tool to discriminate against multiply scattered light (acting as a gating mechanism) in order to enhance contrast and to improve tissue imaging resolution. Moreover, the intrinsic tissue polarimetry characteristics contain a wealth of morphological and functional information of potential biomedical importance. However, in a complex random medium-like tissue, numerous complexities due to multiple scattering and simultaneous occurrences of many scattering and polarization events present formidable challenges both in terms of accurate measurements and in terms of analysis of the tissue polarimetry signal. In order to realize the potential of the polarimetric approaches for tissue imaging and characterization/diagnosis, a number of researchers are thus pursuing innovative solutions to these challenges. In this review paper, we summarize these and other issues pertinent to the polarized light methodologies in tissues. Specifically, we discuss polarized light basics, Stokes-Muller formalism, methods of polarization measurements, polarized light modeling in turbid media, applications to tissue imaging, inverse analysis for polarimetric results quantification, applications to quantitative tissue assessment, etc.

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

PubMed Central

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

2016-01-01

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

A facile and sensitive method for quantification of cyclic nucleotide monophosphates in mammalian organs: basal levels of eight cNMPs and identification of 2',3'-cIMP.

PubMed

Jia, Xin; Fontaine, Benjamin M; Strobel, Fred; Weinert, Emily E

2014-12-12

A sensitive, versatile and economical method to extract and quantify cyclic nucleotide monophosphates (cNMPs) using LC-MS/MS, including both 3',5'-cNMPs and 2',3'-cNMPs, in mammalian tissues and cellular systems has been developed. Problems, such as matrix effects from complex biological samples, are addressed and have been optimized. This protocol allows for comparison of multiple cNMPs in the same system and was used to examine the relationship between tissue levels of cNMPs in a panel of rat organs. In addition, the study reports the first identification and quantification of 2',3'-cIMP. The developed method will allow for quantification of cNMPs levels in cells and tissues with varying disease states, which will provide insight into the role(s) and interplay of cNMP signalling pathways.

Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC-MSE.

PubMed

Creskey, Marybeth C; Li, Changgui; Wang, Junzhi; Girard, Michel; Lorbetskie, Barry; Gravel, Caroline; Farnsworth, Aaron; Li, Xuguang; Smith, Daryl G S; Cyr, Terry D

2012-07-06

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 μg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS(E), a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current

Evaluation of reference genes for insect olfaction studies.

PubMed

Omondi, Bonaventure Aman; Latorre-Estivalis, Jose Manuel; Rocha Oliveira, Ivana Helena; Ignell, Rickard; Lorenzo, Marcelo Gustavo

2015-04-22

Quantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of 'housekeeping genes', involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori. We evaluated eight potential reference genes for their stability as internal controls for RT-qPCR studies of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. The set of genes included were: α-tubulin; β-actin; Glyceraldehyde-3-phosphate dehydrogenase; Eukaryotic initiation factor 1A; Glutathione-S-transferase; Serine protease; Succinate dehydrogenase; and Glucose-6-phosphate dehydrogenase. Five experimental conditions, including changes in age,developmental stage and feeding status were tested in both sexes. We show that the evaluation of candidate reference genes is necessary for each combination of sex, tissue and physiological condition analyzed in order to avoid inconsistent results and conclusions. Although, Normfinder and geNorm software yielded different results between males and females, five genes (SDH, Tub, GAPDH, Act and G6PDH) appeared in the first positions in all rankings obtained. By using gene expression data of a single olfactory coreceptor gene as an example, we demonstrated the extent of changes expected using different internal standards. This work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, we show that particular

Image-based computational quantification and visualization of genetic alterations and tumour heterogeneity

PubMed Central

Zhong, Qing; Rüschoff, Jan H.; Guo, Tiannan; Gabrani, Maria; Schüffler, Peter J.; Rechsteiner, Markus; Liu, Yansheng; Fuchs, Thomas J.; Rupp, Niels J.; Fankhauser, Christian; Buhmann, Joachim M.; Perner, Sven; Poyet, Cédric; Blattner, Miriam; Soldini, Davide; Moch, Holger; Rubin, Mark A.; Noske, Aurelia; Rüschoff, Josef; Haffner, Michael C.; Jochum, Wolfram; Wild, Peter J.

2016-01-01

Recent large-scale genome analyses of human tissue samples have uncovered a high degree of genetic alterations and tumour heterogeneity in most tumour entities, independent of morphological phenotypes and histopathological characteristics. Assessment of genetic copy-number variation (CNV) and tumour heterogeneity by fluorescence in situ hybridization (ISH) provides additional tissue morphology at single-cell resolution, but it is labour intensive with limited throughput and high inter-observer variability. We present an integrative method combining bright-field dual-colour chromogenic and silver ISH assays with an image-based computational workflow (ISHProfiler), for accurate detection of molecular signals, high-throughput evaluation of CNV, expressive visualization of multi-level heterogeneity (cellular, inter- and intra-tumour heterogeneity), and objective quantification of heterogeneous genetic deletions (PTEN) and amplifications (19q12, HER2) in diverse human tumours (prostate, endometrial, ovarian and gastric), using various tissue sizes and different scanners, with unprecedented throughput and reproducibility. PMID:27052161

Image-based computational quantification and visualization of genetic alterations and tumour heterogeneity.

PubMed

Zhong, Qing; Rüschoff, Jan H; Guo, Tiannan; Gabrani, Maria; Schüffler, Peter J; Rechsteiner, Markus; Liu, Yansheng; Fuchs, Thomas J; Rupp, Niels J; Fankhauser, Christian; Buhmann, Joachim M; Perner, Sven; Poyet, Cédric; Blattner, Miriam; Soldini, Davide; Moch, Holger; Rubin, Mark A; Noske, Aurelia; Rüschoff, Josef; Haffner, Michael C; Jochum, Wolfram; Wild, Peter J

2016-04-07

Recent large-scale genome analyses of human tissue samples have uncovered a high degree of genetic alterations and tumour heterogeneity in most tumour entities, independent of morphological phenotypes and histopathological characteristics. Assessment of genetic copy-number variation (CNV) and tumour heterogeneity by fluorescence in situ hybridization (ISH) provides additional tissue morphology at single-cell resolution, but it is labour intensive with limited throughput and high inter-observer variability. We present an integrative method combining bright-field dual-colour chromogenic and silver ISH assays with an image-based computational workflow (ISHProfiler), for accurate detection of molecular signals, high-throughput evaluation of CNV, expressive visualization of multi-level heterogeneity (cellular, inter- and intra-tumour heterogeneity), and objective quantification of heterogeneous genetic deletions (PTEN) and amplifications (19q12, HER2) in diverse human tumours (prostate, endometrial, ovarian and gastric), using various tissue sizes and different scanners, with unprecedented throughput and reproducibility.

Multi-laboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil

USDA-ARS?s Scientific Manuscript database

Accurate identification and quantification of Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, within root tissue and soil are important tasks. Several quantitative PCR (qPCR) assays have been developed but there are no reports comparing their use in sensitive and specific...

Quantification of 11C-Laniquidar Kinetics in the Brain.

PubMed

Froklage, Femke E; Boellaard, Ronald; Bakker, Esther; Hendrikse, N Harry; Reijneveld, Jaap C; Schuit, Robert C; Windhorst, Albert D; Schober, Patrick; van Berckel, Bart N M; Lammertsma, Adriaan A; Postnov, Andrey

2015-11-01

Overexpression of the multidrug efflux transport P-glycoprotein may play an important role in pharmacoresistance. (11)C-laniquidar is a newly developed tracer of P-glycoprotein expression. The aim of this study was to develop a pharmacokinetic model for quantification of (11)C-laniquidar uptake and to assess its test-retest variability. Two (test-retest) dynamic (11)C-laniquidar PET scans were obtained in 8 healthy subjects. Plasma input functions were obtained using online arterial blood sampling with metabolite corrections derived from manual samples. Coregistered T1 MR images were used for region-of-interest definition. Time-activity curves were analyzed using various plasma input compartmental models. (11)C-laniquidar was metabolized rapidly, with a parent plasma fraction of 50% at 10 min after tracer injection. In addition, the first-pass extraction of (11)C-laniquidar was low. (11)C-laniquidar time-activity curves were best fitted to an irreversible single-tissue compartment (1T1K) model using conventional models. Nevertheless, significantly better fits were obtained using 2 parallel single-tissue compartments, one for parent tracer and the other for labeled metabolites (dual-input model). Robust K1 results were also obtained by fitting the first 5 min of PET data to the 1T1K model, at least when 60-min plasma input data were used. For both models, the test-retest variability of (11)C-laniquidar rate constant for transfer from arterial plasma to tissue (K1) was approximately 19%. The accurate quantification of (11)C-laniquidar kinetics in the brain is hampered by its fast metabolism and the likelihood that labeled metabolites enter the brain. Best fits for the entire 60 min of data were obtained using a dual-input model, accounting for uptake of (11)C-laniquidar and its labeled metabolites. Alternatively, K1 could be obtained from a 5-min scan using a standard 1T1K model. In both cases, the test-retest variability of K1 was approximately 19%. © 2015 by the

Reference genes for reverse transcription quantitative PCR in canine brain tissue.

PubMed

Stassen, Quirine E M; Riemers, Frank M; Reijmerink, Hannah; Leegwater, Peter A J; Penning, Louis C

2015-12-09

In the last decade canine models have been used extensively to study genetic causes of neurological disorders such as epilepsy and Alzheimer's disease and unravel their pathophysiological pathways. Reverse transcription quantitative polymerase chain reaction is a sensitive and inexpensive method to study expression levels of genes involved in disease processes. Accurate normalisation with stably expressed so-called reference genes is crucial for reliable expression analysis. Following the minimum information for publication of quantitative real-time PCR experiments precise guidelines, the expression of ten frequently used reference genes, namely YWHAZ, HMBS, B2M, SDHA, GAPDH, HPRT, RPL13A, RPS5, RPS19 and GUSB was evaluated in seven brain regions (frontal lobe, parietal lobe, occipital lobe, temporal lobe, thalamus, hippocampus and cerebellum) and whole brain of healthy dogs. The stability of expression varied between different brain areas. Using the GeNorm and Normfinder software HMBS, GAPDH and HPRT were the most reliable reference genes for whole brain. Furthermore based on GeNorm calculations it was concluded that as little as two to three reference genes are sufficient to obtain reliable normalisation, irrespective the brain area. Our results amend/extend the limited previously published data on canine brain reference genes. Despite the excellent expression stability of HMBS, GAPDH and HRPT, the evaluation of expression stability of reference genes must be a standard and integral part of experimental design and subsequent data analysis.

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

PubMed

Meyer, Marjolaine D; Terry, Leon A

2008-08-27

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

Quantification of methionine and selenomethionine in biological samples using multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS).

PubMed

Vu, Dai Long; Ranglová, Karolína; Hájek, Jan; Hrouzek, Pavel

2018-05-01

Quantification of selenated amino-acids currently relies on methods employing inductively coupled plasma mass spectrometry (ICP-MS). Although very accurate, these methods do not allow the simultaneous determination of standard amino-acids, hampering the comparison of the content of selenated versus non-selenated species such as methionine (Met) and selenomethionine (SeMet). This paper reports two approaches for the simultaneous quantification of Met and SeMet. In the first approach, standard enzymatic hydrolysis employing Protease XIV was applied for the preparation of samples. The second approach utilized methanesulfonic acid (MA) for the hydrolysis of samples, either in a reflux system or in a microwave oven, followed by derivatization with diethyl ethoxymethylenemalonate. The prepared samples were then analyzed by multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS). Both approaches provided platforms for the accurate determination of selenium/sulfur substitution rate in Met. Moreover the second approach also provided accurate simultaneous quantification of Met and SeMet with a low limit of detection, low limit of quantification and wide linearity range, comparable to the commonly used gas chromatography mass spectrometry (GC-MS) method or ICP-MS. The novel method was validated using certified reference material in conjunction with the GC-MS reference method. Copyright © 2018. Published by Elsevier B.V.

Polarization-sensitive optical coherence tomography-based imaging, parameterization, and quantification of human cartilage degeneration

NASA Astrophysics Data System (ADS)

Brill, Nicolai; Wirtz, Mathias; Merhof, Dorit; Tingart, Markus; Jahr, Holger; Truhn, Daniel; Schmitt, Robert; Nebelung, Sven

2016-07-01

Polarization-sensitive optical coherence tomography (PS-OCT) is a light-based, high-resolution, real-time, noninvasive, and nondestructive imaging modality yielding quasimicroscopic cross-sectional images of cartilage. As yet, comprehensive parameterization and quantification of birefringence and tissue properties have not been performed on human cartilage. PS-OCT and algorithm-based image analysis were used to objectively grade human cartilage degeneration in terms of surface irregularity, tissue homogeneity, signal attenuation, as well as birefringence coefficient and band width, height, depth, and number. Degeneration-dependent changes were noted for the former three parameters exclusively, thereby questioning the diagnostic value of PS-OCT in the assessment of human cartilage degeneration.

Magnetic Resonance Spectroscopy: An Objective Technique for the Quantification of Prostate Cancer Pathologies

DTIC Science & Technology

2005-02-01

Holshouser BA, Burley T, Ashwal S . Predicting neuropsychologic outcome after traumatic brain injury in children . Pediatr Neurol 2003;28(2):104-114. 5...breast cancer tissue. NMR Biomed 2002;15(5):327-337. 25. Ala- Korpela M, Posio P, Mattila S , Korhonen A, Williams SR. Absolute quantification of...STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author( s ) and

Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone

PubMed Central

Zehentmeier, Sandra; Cseresnyes, Zoltan; Escribano Navarro, Juan; Niesner, Raluca A.; Hauser, Anja E.

2015-01-01

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data. PMID:25938636

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

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

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

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 weremore » 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.« less

Quantification of regional fat volume in rat MRI

NASA Astrophysics Data System (ADS)

Sacha, Jaroslaw P.; Cockman, Michael D.; Dufresne, Thomas E.; Trokhan, Darren

2003-05-01

Multiple initiatives in the pharmaceutical and beauty care industries are directed at identifying therapies for weight management. Body composition measurements are critical for such initiatives. Imaging technologies that can be used to measure body composition noninvasively include DXA (dual energy x-ray absorptiometry) and MRI (magnetic resonance imaging). Unlike other approaches, MRI provides the ability to perform localized measurements of fat distribution. Several factors complicate the automatic delineation of fat regions and quantification of fat volumes. These include motion artifacts, field non-uniformity, brightness and contrast variations, chemical shift misregistration, and ambiguity in delineating anatomical structures. We have developed an approach to deal practically with those challenges. The approach is implemented in a package, the Fat Volume Tool, for automatic detection of fat tissue in MR images of the rat abdomen, including automatic discrimination between abdominal and subcutaneous regions. We suppress motion artifacts using masking based on detection of implicit landmarks in the images. Adaptive object extraction is used to compensate for intensity variations. This approach enables us to perform fat tissue detection and quantification in a fully automated manner. The package can also operate in manual mode, which can be used for verification of the automatic analysis or for performing supervised segmentation. In supervised segmentation, the operator has the ability to interact with the automatic segmentation procedures to touch-up or completely overwrite intermediate segmentation steps. The operator's interventions steer the automatic segmentation steps that follow. This improves the efficiency and quality of the final segmentation. Semi-automatic segmentation tools (interactive region growing, live-wire, etc.) improve both the accuracy and throughput of the operator when working in manual mode. The quality of automatic segmentation has been

Quantification of Cardiomyocyte Alignment from Three-Dimensional (3D) Confocal Microscopy of Engineered Tissue.

PubMed

Kowalski, William J; Yuan, Fangping; Nakane, Takeichiro; Masumoto, Hidetoshi; Dwenger, Marc; Ye, Fei; Tinney, Joseph P; Keller, Bradley B

2017-08-01

Biological tissues have complex, three-dimensional (3D) organizations of cells and matrix factors that provide the architecture necessary to meet morphogenic and functional demands. Disordered cell alignment is associated with congenital heart disease, cardiomyopathy, and neurodegenerative diseases and repairing or replacing these tissues using engineered constructs may improve regenerative capacity. However, optimizing cell alignment within engineered tissues requires quantitative 3D data on cell orientations and both efficient and validated processing algorithms. We developed an automated method to measure local 3D orientations based on structure tensor analysis and incorporated an adaptive subregion size to account for multiple scales. Our method calculates the statistical concentration parameter, κ, to quantify alignment, as well as the traditional orientational order parameter. We validated our method using synthetic images and accurately measured principal axis and concentration. We then applied our method to confocal stacks of cleared, whole-mount engineered cardiac tissues generated from human-induced pluripotent stem cells or embryonic chick cardiac cells and quantified cardiomyocyte alignment. We found significant differences in alignment based on cellular composition and tissue geometry. These results from our synthetic images and confocal data demonstrate the efficiency and accuracy of our method to measure alignment in 3D tissues.

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.

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.

X-ray Phase Contrast Imaging of Calcified Tissue and Biomaterial Structure in Bioreactor Engineered Tissues

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

Appel, Alyssa A.; Larson, Jeffery C.; Garson, III, Alfred B.

2014-11-04

Tissues engineered in bioreactor systems have been used clinically to replace damaged tissues and organs. In addition, these systems are under continued development for many tissue engineering applications. The ability to quantitatively assess material structure and tissue formation is critical for evaluating bioreactor efficacy and for preimplantation assessment of tissue quality. These techniques allow for the nondestructive and longitudinal monitoring of large engineered tissues within the bioreactor systems and will be essential for the translation of these strategies to viable clinical therapies. X-ray Phase Contrast (XPC) imaging techniques have shown tremendous promise for a number of biomedical applications owing tomore » their ability to provide image contrast based on multiple X-ray properties, including absorption, refraction, and scatter. In this research, mesenchymal stem cell-seeded alginate hydrogels were prepared and cultured under osteogenic conditions in a perfusion bioreactor. The constructs were imaged at various time points using XPC microcomputed tomography (µCT). Imaging was performed with systems using both synchrotron- and tube-based X-ray sources. XPC µCT allowed for simultaneous three-dimensional (3D) quantification of hydrogel size and mineralization, as well as spatial information on hydrogel structure and mineralization. Samples were processed for histological evaluation and XPC showed similar features to histology and quantitative analysis consistent with the histomorphometry. Furthermore, these results provide evidence of the significant potential of techniques based on XPC for noninvasive 3D imaging engineered tissues grown in bioreactors.« less

A Facile and Sensitive Method for Quantification of Cyclic Nucleotide Monophosphates in Mammalian Organs: Basal Levels of Eight cNMPs and Identification of 2',3'-cIMP

PubMed Central

Jia, Xin; Fontaine, Benjamin M.; Strobel, Fred; Weinert, Emily E.

2014-01-01

A sensitive, versatile and economical method to extract and quantify cyclic nucleotide monophosphates (cNMPs) using LC-MS/MS, including both 3',5'-cNMPs and 2',3'-cNMPs, in mammalian tissues and cellular systems has been developed. Problems, such as matrix effects from complex biological samples, are addressed and have been optimized. This protocol allows for comparison of multiple cNMPs in the same system and was used to examine the relationship between tissue levels of cNMPs in a panel of rat organs. In addition, the study reports the first identification and quantification of 2',3'-cIMP. The developed method will allow for quantification of cNMPs levels in cells and tissues with varying disease states, which will provide insight into the role(s) and interplay of cNMP signalling pathways. PMID:25513747

Whole-Body Computed Tomography-Based Body Mass and Body Fat Quantification: A Comparison to Hydrostatic Weighing and Air Displacement Plethysmography.

PubMed

Gibby, Jacob T; Njeru, Dennis K; Cvetko, Steve T; Heiny, Eric L; Creer, Andrew R; Gibby, Wendell A

We correlate and evaluate the accuracy of accepted anthropometric methods of percent body fat (%BF) quantification, namely, hydrostatic weighing (HW) and air displacement plethysmography (ADP), to 2 automatic adipose tissue quantification methods using computed tomography (CT). Twenty volunteer subjects (14 men, 6 women) received head-to-toe CT scans. Hydrostatic weighing and ADP were obtained from 17 and 12 subjects, respectively. The CT data underwent conversion using 2 separate algorithms, namely, the Schneider method and the Beam method, to convert Hounsfield units to their respective tissue densities. The overall mass and %BF of both methods were compared with HW and ADP. When comparing ADP to CT data using the Schneider method and Beam method, correlations were r = 0.9806 and 0.9804, respectively. Paired t tests indicated there were no statistically significant biases. Additionally, observed average differences in %BF between ADP and the Schneider method and the Beam method were 0.38% and 0.77%, respectively. The %BF measured from ADP, the Schneider method, and the Beam method all had significantly higher mean differences when compared with HW (3.05%, 2.32%, and 1.94%, respectively). We have shown that total body mass correlates remarkably well with both the Schneider method and Beam method of mass quantification. Furthermore, %BF calculated with the Schneider method and Beam method CT algorithms correlates remarkably well with ADP. The application of these CT algorithms have utility in further research to accurately stratify risk factors with periorgan, visceral, and subcutaneous types of adipose tissue, and has the potential for significant clinical application.

Quantification of change in vocal fold tissue stiffness relative to depth of artificial damage.

PubMed

Rohlfs, Anna-Katharina; Schmolke, Sebastian; Clauditz, Till; Hess, Markus; Müller, Frank; Püschel, Klaus; Roemer, Frank W; Schumacher, Udo; Goodyer, Eric

2017-10-01

To quantify changes in the biomechanical properties of human excised vocal folds with defined artificial damage. The linear skin rheometer (LSR) was used to obtain a series of rheological measurements of shear modulus from the surface of 30 human cadaver vocal folds. The tissue samples were initially measured in a native condition and then following varying intensities of thermal damage. Histological examination of each vocal fold was used to determine the depth of artificial alteration. The measured changes in stiffness were correlated with the depth of cell damage. For vocal folds in a pre-damage state the shear modulus values ranged from 537 Pa to 1,651 Pa (female) and from 583 Pa to 1,193 Pa (male). With increasing depth of damage from the intermediate layer of the lamina propria (LP), tissue stiffness increased consistently (compared with native values) following application of thermal damage to the vocal folds. The measurement showed an increase of tissue stiffness when the depth of tissue damage was extending from the intermediate LP layer downwards. Changes in the elastic characteristics of human vocal fold tissue following damage at defined depths were demonstrated in an in vitro experiment. In future, reproducible in vivo measurements of elastic vocal fold tissue alterations may enable phonosurgeons to infer the extent of subepithelial damage from changes in surface elasticity.

New approach for the quantification of processed animal proteins in feed using light microscopy.

PubMed

Veys, P; Baeten, V

2010-07-01

A revision of European Union's total feed ban on animal proteins in feed will need robust quantification methods, especially for control analyses, if tolerance levels are to be introduced, as for fishmeal in ruminant feed. In 2006, a study conducted by the Community Reference Laboratory for Animal Proteins in feedstuffs (CRL-AP) demonstrated the deficiency of the official quantification method based on light microscopy. The study concluded that the method had to be revised. This paper puts forward an improved quantification method based on three elements: (1) the preparation of permanent slides with an optical adhesive preserving all morphological markers of bones necessary for accurate identification and precision counting; (2) the use of a counting grid eyepiece reticle; and (3) new definitions for correction factors for the estimated portions of animal particles in the sediment. This revised quantification method was tested on feeds adulterated at different levels with bovine meat and bone meal (MBM) and fishmeal, and it proved to be effortless to apply. The results obtained were very close to the expected values of contamination levels for both types of adulteration (MBM or fishmeal). Calculated values were not only replicable, but also reproducible. The advantages of the new approach, including the benefits of the optical adhesive used for permanent slide mounting and the experimental conditions that need to be met to implement the new method correctly, are discussed.

EDC Cross-linking of Decellularized Tissue: A Promising Approach?

PubMed

Lehmann, Nadine; Christ, Torsten; Daugs, Aila; Bloch, Oliver; Holinski, Sebastian

2017-07-01

Decellularization of xenogenous cardiovascular structures is a promising approach to create scaffolds for tissue engineering. Unfortunately, handling and pliability of the unfixed tissue is challenging. N-(3-dimethylaminopropyl)-N9-ethylcarbodiimide (EDC) is an alternative cross-linking agent to glutaraldehyde (GA). Applied in native tissue, it provides biocompatibility and shows no potential for calcification. In addition, EDC can be used to link growth factors (GFs) to tissue scaffolds after decellularization. EDC cross-linking could thereby help to improve decellularized tissue without the toxicity of GA. Porcine aortic wall tissue specimens (TS) were decellularized, treated with EDC, and coated with fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF). Afterward, TS were subcutaneously implanted in 36 Lewis rats along with one decellularized TS without EDC treatment. After 2, 4, and 6 weeks TS were explanted from 12 rats, respectively. Vital cells were evaluated by RNA quantification, general cellular infiltration by hematoxylin and eosin staining (H&E), macrophage infiltration by CD68 staining, calcification by Von-Kossa staining, and tissue degradation by measurement of TS thickness. Quantification of vital cells showed reduced reseeding of EDC-treated TS compared to noncross-linked TS after 2 (p < 0.05) and 4 weeks (p < 0.05), while after 6 weeks only EDC+VEGF showed fewer viable cells (p < 0.01). Histological evaluation confirmed a reduced infiltration of EDC-treated TS. Macrophage infiltration decreased in all groups from 2 to 6 weeks, with the smallest population in EDC+VEGF-treated TS (p > 0.05). In EDC+FGF-treated TS, macrophages were reduced after 2 weeks compared to noncross-linked TS (p < 0.05), while after 4 and 6 weeks no significant difference was found (p > 0.05). Von-Kossa staining revealed no calcification in any of the specimens. Thickness of noncross-linked and EDC+FGF-treated TS was not

New class of radioenzymatic assay for the quantification of p-tyramine and phenylethylamine

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

Henry, D.P.; Van Huysse, J.W.; Bowsher, R.R.

Radioenzymatic assays are widely used for the quantification of a number of biogenic amines. All previous procedures have utilized methyltransferases derived from mammalian tissues. In this assay for the quantification of the trace aralkylamines, p-tyramine (p-tym) and phenylethylamine (PEA), an enzyme, tyramine N-methyltransferase isolated from sprouted barley roots was used. The enzyme was specific for phenylethylamines. Of 26 structurally-related compounds, only p-tym, PEA, m-tym and amphetamine were substrates in vitro. Theoretic maximal methylation of substrates occurred at 10-20/sup 0/C. When TLC was used to separate the radiolabeled reaction products, a specific method was developed for p-tym and PEA. The assaymore » had a sensitivity of 0.8 and 2.8 pg/tube with a C.V. < 5% and was applicable to human plasma and urine. Assay throughput is similar to that of other TLC based radioenzymatic assays.« less

In-line multipoint near-infrared spectroscopy for moisture content quantification during freeze-drying.

PubMed

Kauppinen, Ari; Toiviainen, Maunu; Korhonen, Ossi; Aaltonen, Jaakko; Järvinen, Kristiina; Paaso, Janne; Juuti, Mikko; Ketolainen, Jarkko

2013-02-19

During the past decade, near-infrared (NIR) spectroscopy has been applied for in-line moisture content quantification during a freeze-drying process. However, NIR has been used as a single-vial technique and thus is not representative of the entire batch. This has been considered as one of the main barriers for NIR spectroscopy becoming widely used in process analytical technology (PAT) for freeze-drying. Clearly it would be essential to monitor samples that reliably represent the whole batch. The present study evaluated multipoint NIR spectroscopy for in-line moisture content quantification during a freeze-drying process. Aqueous sucrose solutions were used as model formulations. NIR data was calibrated to predict the moisture content using partial least-squares (PLS) regression with Karl Fischer titration being used as a reference method. PLS calibrations resulted in root-mean-square error of prediction (RMSEP) values lower than 0.13%. Three noncontact, diffuse reflectance NIR probe heads were positioned on the freeze-dryer shelf to measure the moisture content in a noninvasive manner, through the side of the glass vials. The results showed that the detection of unequal sublimation rates within a freeze-dryer shelf was possible with the multipoint NIR system in use. Furthermore, in-line moisture content quantification was reliable especially toward the end of the process. These findings indicate that the use of multipoint NIR spectroscopy can achieve representative quantification of moisture content and hence a drying end point determination to a desired residual moisture level.

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative.

PubMed

Loibner, Martina; Buzina, Walter; Viertler, Christian; Groelz, Daniel; Hausleitner, Anja; Siaulyte, Gintare; Kufferath, Iris; Kölli, Bettina; Zatloukal, Kurt

2016-01-01

Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment.

Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.

PubMed

Li, Xin; Ben-Dov, Iddo Z; Mauro, Maurizio; Williams, Zev

2015-05-06

RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.

Uncertainty quantification for constitutive model calibration of brain tissue.

PubMed

Brewick, Patrick T; Teferra, Kirubel

2018-05-31

The results of a study comparing model calibration techniques for Ogden's constitutive model that describes the hyperelastic behavior of brain tissue are presented. One and two-term Ogden models are fit to two different sets of stress-strain experimental data for brain tissue using both least squares optimization and Bayesian estimation. For the Bayesian estimation, the joint posterior distribution of the constitutive parameters is calculated by employing Hamiltonian Monte Carlo (HMC) sampling, a type of Markov Chain Monte Carlo method. The HMC method is enriched in this work to intrinsically enforce the Drucker stability criterion by formulating a nonlinear parameter constraint function, which ensures the constitutive model produces physically meaningful results. Through application of the nested sampling technique, 95% confidence bounds on the constitutive model parameters are identified, and these bounds are then propagated through the constitutive model to produce the resultant bounds on the stress-strain response. The behavior of the model calibration procedures and the effect of the characteristics of the experimental data are extensively evaluated. It is demonstrated that increasing model complexity (i.e., adding an additional term in the Ogden model) improves the accuracy of the best-fit set of parameters while also increasing the uncertainty via the widening of the confidence bounds of the calibrated parameters. Despite some similarity between the two data sets, the resulting distributions are noticeably different, highlighting the sensitivity of the calibration procedures to the characteristics of the data. For example, the amount of uncertainty reported on the experimental data plays an essential role in how data points are weighted during the calibration, and this significantly affects how the parameters are calibrated when combining experimental data sets from disparate sources. Published by Elsevier Ltd.

Quantification of protein expression in cells and cellular subcompartments on immunohistochemical sections using a computer supported image analysis system.

PubMed

Braun, Martin; Kirsten, Robert; Rupp, Niels J; Moch, Holger; Fend, Falko; Wernert, Nicolas; Kristiansen, Glen; Perner, Sven

2013-05-01

Quantification of protein expression based on immunohistochemistry (IHC) is an important step for translational research and clinical routine. Several manual ('eyeballing') scoring systems are used in order to semi-quantify protein expression based on chromogenic intensities and distribution patterns. However, manual scoring systems are time-consuming and subject to significant intra- and interobserver variability. The aim of our study was to explore, whether new image analysis software proves to be sufficient as an alternative tool to quantify protein expression. For IHC experiments, one nucleus specific marker (i.e., ERG antibody), one cytoplasmic specific marker (i.e., SLC45A3 antibody), and one marker expressed in both compartments (i.e., TMPRSS2 antibody) were chosen. Stainings were applied on TMAs, containing tumor material of 630 prostate cancer patients. A pathologist visually quantified all IHC stainings in a blinded manner, applying a four-step scoring system. For digital quantification, image analysis software (Tissue Studio v.2.1, Definiens AG, Munich, Germany) was applied to obtain a continuous spectrum of average staining intensity. For each of the three antibodies we found a strong correlation of the manual protein expression score and the score of the image analysis software. Spearman's rank correlation coefficient was 0.94, 0.92, and 0.90 for ERG, SLC45A3, and TMPRSS2, respectively (p⟨0.01). Our data suggest that the image analysis software Tissue Studio is a powerful tool for quantification of protein expression in IHC stainings. Further, since the digital analysis is precise and reproducible, computer supported protein quantification might help to overcome intra- and interobserver variability and increase objectivity of IHC based protein assessment.

Statistical image quantification toward optimal scan fusion and change quantification

NASA Astrophysics Data System (ADS)

Potesil, Vaclav; Zhou, Xiang Sean

2007-03-01

Recent advance of imaging technology has brought new challenges and opportunities for automatic and quantitative analysis of medical images. With broader accessibility of more imaging modalities for more patients, fusion of modalities/scans from one time point and longitudinal analysis of changes across time points have become the two most critical differentiators to support more informed, more reliable and more reproducible diagnosis and therapy decisions. Unfortunately, scan fusion and longitudinal analysis are both inherently plagued with increased levels of statistical errors. A lack of comprehensive analysis by imaging scientists and a lack of full awareness by physicians pose potential risks in clinical practice. In this paper, we discuss several key error factors affecting imaging quantification, studying their interactions, and introducing a simulation strategy to establish general error bounds for change quantification across time. We quantitatively show that image resolution, voxel anisotropy, lesion size, eccentricity, and orientation are all contributing factors to quantification error; and there is an intricate relationship between voxel anisotropy and lesion shape in affecting quantification error. Specifically, when two or more scans are to be fused at feature level, optimal linear fusion analysis reveals that scans with voxel anisotropy aligned with lesion elongation should receive a higher weight than other scans. As a result of such optimal linear fusion, we will achieve a lower variance than naïve averaging. Simulated experiments are used to validate theoretical predictions. Future work based on the proposed simulation methods may lead to general guidelines and error lower bounds for quantitative image analysis and change detection.

Semi-automated quantification and neuroanatomical mapping of heterogeneous cell populations.

PubMed

Mendez, Oscar A; Potter, Colin J; Valdez, Michael; Bello, Thomas; Trouard, Theodore P; Koshy, Anita A

2018-07-15

Our group studies the interactions between cells of the brain and the neurotropic parasite Toxoplasma gondii. Using an in vivo system that allows us to permanently mark and identify brain cells injected with Toxoplasma protein, we have identified that Toxoplasma-injected neurons (TINs) are heterogeneously distributed throughout the brain. Unfortunately, standard methods to quantify and map heterogeneous cell populations onto a reference brain atlas are time consuming and prone to user bias. We developed a novel MATLAB-based semi-automated quantification and mapping program to allow the rapid and consistent mapping of heterogeneously distributed cells on to the Allen Institute Mouse Brain Atlas. The system uses two-threshold background subtraction to identify and quantify cells of interest. We demonstrate that we reliably quantify and neuroanatomically localize TINs with low intra- or inter-observer variability. In a follow up experiment, we show that specific regions of the mouse brain are enriched with TINs. The procedure we use takes advantage of simple immunohistochemistry labeling techniques, use of a standard microscope with a motorized stage, and low cost computing that can be readily obtained at a research institute. To our knowledge there is no other program that uses such readily available techniques and equipment for mapping heterogeneous populations of cells across the whole mouse brain. The quantification method described here allows reliable visualization, quantification, and mapping of heterogeneous cell populations in immunolabeled sections across whole mouse brains. Copyright © 2018 Elsevier B.V. All rights reserved.

Thermal and molecular investigation of laser tissue welding

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

Small, W., IV

1998-06-01

Despite the growing number of successful animal and human trials, the exact mechanisms of laser tissue welding remain unknown. Furthermore, the effects of laser heating on tissue on the molecular scale are not fully understood. To address these issues, a multi-front attack oil both extrinsic (solder/patch mediated) and intrinsic (laser only) tissue welding was launched using two-color infrared thermometry, computer modeling, weld strength assessment, biochemical assays, and vibrational spectroscopy. The coupling of experimentally measured surface temperatures with the predictive numerical simulations provided insight into the sub-surface dynamics of the laser tissue welding process. Quantification of the acute strength of themore » welds following the welding procedure enabled comparison among trials during an experiment, with previous experiments, and with other studies in the literature. The acute weld integrity also provided an indication of tile probability of long-term success. Molecular effects induced In the tissue by laser irradiation were investigated by measuring tile concentrations of specific collagen covalent crosslinks and characterizing the Fourier-Transform infrared (FTIR) spectra before and after the laser exposure.« less

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

PubMed

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

2016-01-01

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

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

PubMed

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

2014-08-26

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

Applicability of plasmid calibrant pTC1507 in quantification of TC1507 maize: an interlaboratory study.

PubMed

Meng, Yanan; Liu, Xin; Wang, Shu; Zhang, Dabing; Yang, Litao

2012-01-11

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of DNA plasmids as calibrants is becoming essential for the practical quantification of GMOs. This study reports the construction of plasmid pTC1507 for a quantification assay of genetically modified (GM) maize TC1507 and the collaborative ring trial in international validation of its applicability as a plasmid calibrant. pTC1507 includes one event-specific sequence of TC1507 maize and one unique sequence of maize endogenous gene zSSIIb. A total of eight GMO detection laboratories worldwide were invited to join the validation process, and test results were returned from all eight participants. Statistical analysis of the returned results showed that real-time PCR assays using pTC1507 as calibrant in both GM event-specific and endogenous gene quantifications had high PCR efficiency (ranging from 0.80 to 1.15) and good linearity (ranging from 0.9921 to 0.9998). In a quantification assay of five blind samples, the bias between the test values and true values ranged from 2.6 to 24.9%. All results indicated that the developed pTC1507 plasmid is applicable for the quantitative analysis of TC1507 maize and can be used as a suitable substitute for dried powder certified reference materials (CRMs).

Fluorescent quantification of melanin.

PubMed

Fernandes, Bruno; Matamá, Teresa; Guimarães, Diana; Gomes, Andreia; Cavaco-Paulo, Artur

2016-11-01

Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non-melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Therefore, fluorescence spectroscopy is the best method for melanin quantification as it proved to be highly specific and accurate, detecting even small variations in the synthesis of melanin. This method can also be applied to the quantification of melanin in more complex biological matrices like zebrafish embryos and human hair. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Phase Contrast Microscopy Analysis of Breast Tissue

PubMed Central

Wells, Wendy A.; Wang, Xin; Daghlian, Charles P.; Paulsen, Keith D.; Pogue, Brian W.

2010-01-01

OBJECTIVE To assess how optical scatter properties in breast tissue, as measured by phase contrast microscopy and interpreted pathophysiologically, might be exploited as a diagnostic tool to differentiate cancer from benign tissue. STUDY DESIGN We evaluated frozen human breast tissue sections of adipose tissue, normal breast parenchyma, benign fibroadenoma tumors and noninvasive and invasive malignant cancers by phase contrast microscopy through quantification of grayscale values, using multiple regions of interest (ROI). Student’s t tests were performed on phase contrast measures across diagnostic categories testing data from individual cases; all ROI data were used as separate measures. RESULTS Stroma demonstrated significantly higher scatter intensity than did epithelium, with lower scattering in tumor-associated stroma as compared with normal or benign-associated stroma. Measures were comparable for invasive and noninvasive malignant tumors but were higher than those found in benign tumors and were lowest in adipose tissue. CONCLUSION Significant differences were found in scatter coefficient properties of epithelium and stroma across diagnostic categories of breast tissue, particularly between benign and malignant-associated stroma. Improved understanding of how scatter properties correlate with morphologic criteria used in routine pathologic diagnoses could have a significant clinical impact as developing optical technology allows macroscopic in situ phase contrast imaging. PMID:19736867

Breast density quantification with cone-beam CT: A post-mortem study

PubMed Central

Johnson, Travis; Ding, Huanjun; Le, Huy Q.; Ducote, Justin L.; Molloi, Sabee

2014-01-01

Forty post-mortem breasts were imaged with a flat-panel based cone-beam x-ray CT system at 50 kVp. The feasibility of breast density quantification has been investigated using standard histogram thresholding and an automatic segmentation method based on the fuzzy c-means algorithm (FCM). The breasts were chemically decomposed into water, lipid, and protein immediately after image acquisition was completed. The percent fibroglandular volume (%FGV) from chemical analysis was used as the gold standard for breast density comparison. Both image-based segmentation techniques showed good precision in breast density quantification with high linear coefficients between the right and left breast of each pair. When comparing with the gold standard using %FGV from chemical analysis, Pearson’s r-values were estimated to be 0.983 and 0.968 for the FCM clustering and the histogram thresholding techniques, respectively. The standard error of the estimate (SEE) was also reduced from 3.92% to 2.45% by applying the automatic clustering technique. The results of the postmortem study suggested that breast tissue can be characterized in terms of water, lipid and protein contents with high accuracy by using chemical analysis, which offers a gold standard for breast density studies comparing different techniques. In the investigated image segmentation techniques, the FCM algorithm had high precision and accuracy in breast density quantification. In comparison to conventional histogram thresholding, it was more efficient and reduced inter-observer variation. PMID:24254317

Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

PubMed Central

Nuriel, Tal; Deeb, Ruba S.; Hajjar, David P.; Gross, Steven S.

2008-01-01

Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells and tissues. Tyrosine nitration is recognized as one of the multiple signaling modalities used by NO-derived species for the regulation of protein structure and function in health and disease. Various methods have been described for the quantification of protein 3-nitrotyrosine residues, and several strategies have been presented toward the goal of proteome-wide identification of protein tyrosine modification sites. This chapter details a useful protocol for the quantification of 3-nitrotyrosine in cells and tissues using high-pressure liquid chromatography with electrochemical detection. Additionally, this chapter describes a novel biotin-tagging strategy for specific enrichment of 3-nitrotyrosine-containing peptides. Application of this strategy, in conjunction with high-throughput MS/MS-based peptide sequencing, is anticipated to fuel efforts in developing comprehensive inventories of nitrosative stress-induced protein-tyrosine modification sites in cells and tissues. PMID:18554526

MR/PET quantification tools: Registration, segmentation, classification, and MR-based attenuation correction

PubMed Central

Fei, Baowei; Yang, Xiaofeng; Nye, Jonathon A.; Aarsvold, John N.; Raghunath, Nivedita; Cervo, Morgan; Stark, Rebecca; Meltzer, Carolyn C.; Votaw, John R.

2012-01-01

Purpose: Combined MR/PET is a relatively new, hybrid imaging modality. A human MR/PET prototype system consisting of a Siemens 3T Trio MR and brain PET insert was installed and tested at our institution. Its present design does not offer measured attenuation correction (AC) using traditional transmission imaging. This study is the development of quantification tools including MR-based AC for quantification in combined MR/PET for brain imaging. Methods: The developed quantification tools include image registration, segmentation, classification, and MR-based AC. These components were integrated into a single scheme for processing MR/PET data. The segmentation method is multiscale and based on the Radon transform of brain MR images. It was developed to segment the skull on T1-weighted MR images. A modified fuzzy C-means classification scheme was developed to classify brain tissue into gray matter, white matter, and cerebrospinal fluid. Classified tissue is assigned an attenuation coefficient so that AC factors can be generated. PET emission data are then reconstructed using a three-dimensional ordered sets expectation maximization method with the MR-based AC map. Ten subjects had separate MR and PET scans. The PET with [11C]PIB was acquired using a high-resolution research tomography (HRRT) PET. MR-based AC was compared with transmission (TX)-based AC on the HRRT. Seventeen volumes of interest were drawn manually on each subject image to compare the PET activities between the MR-based and TX-based AC methods. Results: For skull segmentation, the overlap ratio between our segmented results and the ground truth is 85.2 ± 2.6%. Attenuation correction results from the ten subjects show that the difference between the MR and TX-based methods was <6.5%. Conclusions: MR-based AC compared favorably with conventional transmission-based AC. Quantitative tools including registration, segmentation, classification, and MR-based AC have been developed for use in combined MR

Toward automatic segmentation and quantification of tumor and stroma in whole-slide images of H and E stained rectal carcinomas

NASA Astrophysics Data System (ADS)

Geessink, Oscar G. F.; Baidoshvili, Alexi; Freling, Gerard; Klaase, Joost M.; Slump, Cornelis H.; van der Heijden, Ferdinand

2015-03-01

Visual estimation of tumor and stroma proportions in microscopy images yields a strong, Tumor-(lymph)Node- Metastasis (TNM) classification-independent predictor for patient survival in colorectal cancer. Therefore, it is also a potent (contra)indicator for adjuvant chemotherapy. However, quantification of tumor and stroma through visual estimation is highly subject to intra- and inter-observer variability. The aim of this study is to develop and clinically validate a method for objective quantification of tumor and stroma in standard hematoxylin and eosin (H and E) stained microscopy slides of rectal carcinomas. A tissue segmentation algorithm, based on supervised machine learning and pixel classification, was developed, trained and validated using histological slides that were prepared from surgically excised rectal carcinomas in patients who had not received neoadjuvant chemotherapy and/or radiotherapy. Whole-slide scanning was performed at 20× magnification. A total of 40 images (4 million pixels each) were extracted from 20 whole-slide images at sites showing various relative proportions of tumor and stroma. Experienced pathologists provided detailed annotations for every extracted image. The performance of the algorithm was evaluated using cross-validation by testing on 1 image at a time while using the other 39 images for training. The total classification error of the algorithm was 9.4% (SD = 3.2%). Compared to visual estimation by pathologists, the algorithm was 7.3 times (P = 0.033) more accurate in quantifying tissues, also showing 60% less variability. Automatic tissue quantification was shown to be both reliable and practicable. We ultimately intend to facilitate refined prognostic stratification of (colo)rectal cancer patients and enable better personalized treatment.

How to Make Data a Blessing to Parametric Uncertainty Quantification and Reduction?

NASA Astrophysics Data System (ADS)

Ye, M.; Shi, X.; Curtis, G. P.; Kohler, M.; Wu, J.

2013-12-01

In a Bayesian point of view, probability of model parameters and predictions are conditioned on data used for parameter inference and prediction analysis. It is critical to use appropriate data for quantifying parametric uncertainty and its propagation to model predictions. However, data are always limited and imperfect. When a dataset cannot properly constrain model parameters, it may lead to inaccurate uncertainty quantification. While in this case data appears to be a curse to uncertainty quantification, a comprehensive modeling analysis may help understand the cause and characteristics of parametric uncertainty and thus turns data into a blessing. In this study, we illustrate impacts of data on uncertainty quantification and reduction using an example of surface complexation model (SCM) developed to simulate uranyl (U(VI)) adsorption. The model includes two adsorption sites, referred to as strong and weak sites. The amount of uranium adsorption on these sites determines both the mean arrival time and the long tail of the breakthrough curves. There is one reaction on the weak site but two reactions on the strong site. The unknown parameters include fractions of the total surface site density of the two sites and surface complex formation constants of the three reactions. A total of seven experiments were conducted with different geochemical conditions to estimate these parameters. The experiments with low initial concentration of U(VI) result in a large amount of parametric uncertainty. A modeling analysis shows that it is because the experiments cannot distinguish the relative adsorption affinity of the strong and weak sites on uranium adsorption. Therefore, the experiments with high initial concentration of U(VI) are needed, because in the experiments the strong site is nearly saturated and the weak site can be determined. The experiments with high initial concentration of U(VI) are a blessing to uncertainty quantification, and the experiments with low initial

Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins

PubMed Central

Chen, Yi; Fisher, Kate J.; Lloyd, Mark; Wood, Elizabeth R.; Coppola, Domenico; Siegel, Erin; Shibata, David; Chen, Yian A.; Koomen, John M.

2017-01-01

Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g. Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays. PMID:28808993

Soft tissue modelling with conical springs.

PubMed

Omar, Nadzeri; Zhong, Yongmin; Jazar, Reza N; Subic, Aleksandar; Smith, Julian; Shirinzadeh, Bijan

2015-01-01

This paper presents a new method for real-time modelling soft tissue deformation. It improves the traditional mass-spring model with conical springs to deal with nonlinear mechanical behaviours of soft tissues. A conical spring model is developed to predict soft tissue deformation with reference to deformation patterns. The model parameters are formulated according to tissue deformation patterns and the nonlinear behaviours of soft tissues are modelled with the stiffness variation of conical spring. Experimental results show that the proposed method can describe different tissue deformation patterns using one single equation and also exhibit the typical mechanical behaviours of soft tissues.

[11C]Harmine Binding to Brain Monoamine Oxidase A: Test-Retest Properties and Noninvasive Quantification.

PubMed

Zanderigo, Francesca; D'Agostino, Alexandra E; Joshi, Nandita; Schain, Martin; Kumar, Dileep; Parsey, Ramin V; DeLorenzo, Christine; Mann, J John

2018-02-08

Inhibition of the isoform A of monoamine oxidase (MAO-A), a mitochondrial enzyme catalyzing deamination of monoamine neurotransmitters, is useful in treatment of depression and anxiety disorders. [ 11 C]harmine, a MAO-A PET radioligand, has been used to study mood disorders and antidepressant treatment. However, [ 11 C]harmine binding test-retest characteristics have to date only been partially investigated. Furthermore, since MAO-A is ubiquitously expressed, no reference region is available, thus requiring arterial blood sampling during PET scanning. Here, we investigate [ 11 C]harmine binding measurements test-retest properties; assess effects of using a minimally invasive input function estimation on binding quantification and repeatability; and explore binding potentials estimation using a reference region-free approach. Quantification of [ 11 C]harmine distribution volume (V T ) via kinetic models and graphical analyses was compared based on absolute test-retest percent difference (TRPD), intraclass correlation coefficient (ICC), and identifiability. The optimal procedure was also used with a simultaneously estimated input function in place of the measured curve. Lastly, an approach for binding potentials quantification in absence of a reference region was evaluated. [ 11 C]harmine V T estimates quantified using arterial blood and kinetic modeling showed average absolute TRPD values of 7.7 to 15.6 %, and ICC values between 0.56 and 0.86, across brain regions. Using simultaneous estimation (SIME) of input function resulted in V T estimates close to those obtained using arterial input function (r = 0.951, slope = 1.073, intercept = - 1.037), with numerically but not statistically higher test-retest difference (range 16.6 to 22.0 %), but with overall poor ICC values, between 0.30 and 0.57. Prospective studies using [ 11 C]harmine are possible given its test-retest repeatability when binding is quantified using arterial blood. Results with SIME of

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas)

PubMed Central

Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults. PMID:26998411

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).

PubMed

Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been; Yang, Wei Cheng

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.

Quantitative CT imaging for adipose tissue analysis in mouse model of obesity

NASA Astrophysics Data System (ADS)

Marchadier, A.; Vidal, C.; Tafani, J.-P.; Ordureau, S.; Lédée, R.; Léger, C.

2011-03-01

In obese humans CT imaging is a validated method for follow up studies of adipose tissue distribution and quantification of visceral and subcutaneous fat. Equivalent methods in murine models of obesity are still lacking. Current small animal micro-CT involves long-term X-ray exposure precluding longitudinal studies. We have overcome this limitation by using a human medical CT which allows very fast 3D imaging (2 sec) and minimal radiation exposure. This work presents novel methods fitted to in vivo investigations of mice model of obesity, allowing (i) automated detection of adipose tissue in abdominal regions of interest, (ii) quantification of visceral and subcutaneous fat. For each mouse, 1000 slices (100μm thickness, 160 μm resolution) were acquired in 2 sec using a Toshiba medical CT (135 kV, 400mAs). A Gaussian mixture model of the Hounsfield curve of 2D slices was computed with the Expectation Maximization algorithm. Identification of each Gaussian part allowed the automatic classification of adipose tissue voxels. The abdominal region of interest (umbilical) was automatically detected as the slice showing the highest ratio of the Gaussian proportion between adipose and lean tissues. Segmentation of visceral and subcutaneous fat compartments was achieved with 2D 1/2 level set methods. Our results show that the application of human clinical CT to mice is a promising approach for the study of obesity, allowing valuable comparison between species using the same imaging materials and software analysis.

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2015-04-01

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. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Simple and rapid quantification of serotonin transporter binding using [11C]DASB bolus plus constant infusion.

PubMed

Gryglewski, G; Rischka, L; Philippe, C; Hahn, A; James, G M; Klebermass, E; Hienert, M; Silberbauer, L; Vanicek, T; Kautzky, A; Berroterán-Infante, N; Nics, L; Traub-Weidinger, T; Mitterhauser, M; Wadsak, W; Hacker, M; Kasper, S; Lanzenberger, R

2017-04-01

In-vivo quantification of serotonin transporters (SERT) in human brain has been a mainstay of molecular imaging in the field of neuropsychiatric disorders and helped to explore the underpinnings of several medical conditions, therapeutic and environmental influences. The emergence of PET/MR hybrid systems and the heterogeneity of SERT binding call for the development of efficient methods making the investigation of larger or vulnerable populations with limited scanner time and simultaneous changes in molecular and functional measures possible. We propose [ 11 C]DASB bolus plus constant infusion for these applications and validate it against standard analyses of dynamic PET data. [ 11 C]DASB bolus/infusion optimization was performed on data acquired after [ 11 C]DASB bolus in 8 healthy subjects. Subsequently, 16 subjects underwent one scan using [ 11 C]DASB bolus plus constant infusion with K bol 160-179min and one scan after [ 11 C]DASB bolus for inter-method reliability analysis. Arterial blood sampling and metabolite analysis were performed for all scans. Distribution volumes (V T ) were obtained using Logan plots for bolus scans and ratios between tissue and plasma parent activity for bolus plus infusion scans for different time spans of the scan (V T-70 for 60-70min after start of tracer infusion, V T-90 for 75-90min, V T-120 for 100-120min) in 9 subjects. Omitting blood data, binding potentials (BP ND ) obtained using multilinear reference tissue modeling (MRTM2) and cerebellar gray matter as reference region were compared in 11 subjects. A K bol of 160min was observed to be optimal for rapid equilibration in thalamus and striatum. V T-70 showed good intraclass correlation coefficients (ICCs) of 0.61-0.70 for thalamus, striatal regions and olfactory cortex with bias ≤5.1% compared to bolus scans. ICCs increased to 0.72-0.78 for V T-90 and 0.77-0.93 for V T-120 in these regions. BP ND-90 had negligible bias ≤2.5%, low variability ≤7.9% and ICCs of 0

Effect of Ischemia Duration and Protective Interventions on the Temporal Dynamics of Tissue Composition After Myocardial Infarction

PubMed Central

Fernández-Jiménez, Rodrigo; Galán-Arriola, Carlos; Sánchez-González, Javier; Agüero, Jaume; López-Martín, Gonzalo J.; Gomez-Talavera, Sandra; Garcia-Prieto, Jaime; Benn, Austin; Molina-Iracheta, Antonio; Barreiro-Pérez, Manuel; Martin-García, Ana; García-Lunar, Inés; Pizarro, Gonzalo; Sanz, Javier; Sánchez, Pedro L.; Fuster, Valentin

2017-01-01

Rationale: The impact of cardioprotective strategies and ischemia duration on postischemia/reperfusion (I/R) myocardial tissue composition (edema, myocardium at risk, infarct size, salvage, intramyocardial hemorrhage, and microvascular obstruction) is not well understood. Objective: To study the effect of ischemia duration and protective interventions on the temporal dynamics of myocardial tissue composition in a translational animal model of I/R by the use of state-of-the-art imaging technology. Methods and Results: Four 5-pig groups underwent different I/R protocols: 40-minute I/R (prolonged ischemia, controls), 20-minute I/R (short-duration ischemia), prolonged ischemia preceded by preconditioning, or prolonged ischemia followed by postconditioning. Serial cardiac magnetic resonance (CMR)-based tissue characterization was done in all pigs at baseline and at 120 minutes, day 1, day 4, and day 7 after I/R. Reference myocardium at risk was assessed by multidetector computed tomography during the index coronary occlusion. After the final CMR, hearts were excised and processed for water content quantification and histology. Five additional healthy pigs were euthanized after baseline CMR as reference. Edema formation followed a bimodal pattern in all 40-minute I/R pigs, regardless of cardioprotective strategy and the degree of intramyocardial hemorrhage or microvascular obstruction. The hyperacute edematous wave was ameliorated only in pigs showing cardioprotection (ie, those undergoing short-duration ischemia or preconditioning). In all groups, CMR-measured edema was barely detectable at 24 hours postreperfusion. The deferred healing-related edematous wave was blunted or absent in pigs undergoing preconditioning or short-duration ischemia, respectively. CMR-measured infarct size declined progressively after reperfusion in all groups. CMR-measured myocardial salvage, and the extent of intramyocardial hemorrhage and microvascular obstruction varied dramatically

Exploiting multicompartment effects in triple-echo steady-state T2 mapping for fat fraction quantification.

PubMed

Liu, Dian; Steingoetter, Andreas; Curcic, Jelena; Kozerke, Sebastian

2018-01-01

To investigate and exploit the effect of intravoxel off-resonance compartments in the triple-echo steady-state (TESS) sequence without fat suppression for T 2 mapping and to leverage the results for fat fraction quantification. In multicompartment tissue, where at least one compartment is excited off-resonance, the total signal exhibits periodic modulations as a function of echo time (TE). Simulated multicompartment TESS signals were synthesized at various TEs. Fat emulsion phantoms were prepared and scanned at the same TE combinations using TESS. In vivo knee data were obtained with TESS to validate the simulations. The multicompartment effect was exploited for fat fraction quantification in the stomach by acquiring TESS signals at two TE combinations. Simulated and measured multicompartment signal intensities were in good agreement. Multicompartment effects caused erroneous T 2 offsets, even at low water-fat ratios. The choice of TE caused T 2 variations of as much as 28% in cartilage. The feasibility of fat fraction quantification to monitor the decrease of fat content in the stomach during digestion is demonstrated. Intravoxel off-resonance compartments are a confounding factor for T 2 quantification using TESS, causing errors that are dependent on the TE. At the same time, off-resonance effects may allow for efficient fat fraction mapping using steady-state imaging. Magn Reson Med 79:423-429, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Nondestructive Detection and Quantification of Blueberry Bruising using Near-infrared (NIR) Hyperspectral Reflectance Imaging

NASA Astrophysics Data System (ADS)

Jiang, Yu; Li, Changying; Takeda, Fumiomi

2016-10-01

Currently, blueberry bruising is evaluated by either human visual/tactile inspection or firmness measurement instruments. These methods are destructive, time-consuming, and subjective. The goal of this paper was to develop a non-destructive approach for blueberry bruising detection and quantification. Experiments were conducted on 300 samples of southern highbush blueberry (Camellia, Rebel, and Star) and on 1500 samples of northern highbush blueberry (Bluecrop, Jersey, and Liberty) for hyperspectral imaging analysis, firmness measurement, and human evaluation. An algorithm was developed to automatically calculate a bruise ratio index (ratio of bruised to whole fruit area) for bruise quantification. The spectra of bruised and healthy tissues were statistically separated and the separation was independent of cultivars. Support vector machine (SVM) classification of the spectra from the regions of interest (ROIs) achieved over 94%, 92%, and 96% accuracy on the training set, independent testing set, and combined set, respectively. The statistical results showed that the bruise ratio index was equivalent to the measured firmness but better than the predicted firmness in regard to effectiveness of bruise quantification, and the bruise ratio index had a strong correlation with human assessment (R2 = 0.78 - 0.83). Therefore, the proposed approach and the bruise ratio index are effective to non-destructively detect and quantify blueberry bruising.

Nondestructive Detection and Quantification of Blueberry Bruising using Near-infrared (NIR) Hyperspectral Reflectance Imaging.

PubMed

Jiang, Yu; Li, Changying; Takeda, Fumiomi

2016-10-21

Currently, blueberry bruising is evaluated by either human visual/tactile inspection or firmness measurement instruments. These methods are destructive, time-consuming, and subjective. The goal of this paper was to develop a non-destructive approach for blueberry bruising detection and quantification. Experiments were conducted on 300 samples of southern highbush blueberry (Camellia, Rebel, and Star) and on 1500 samples of northern highbush blueberry (Bluecrop, Jersey, and Liberty) for hyperspectral imaging analysis, firmness measurement, and human evaluation. An algorithm was developed to automatically calculate a bruise ratio index (ratio of bruised to whole fruit area) for bruise quantification. The spectra of bruised and healthy tissues were statistically separated and the separation was independent of cultivars. Support vector machine (SVM) classification of the spectra from the regions of interest (ROIs) achieved over 94%, 92%, and 96% accuracy on the training set, independent testing set, and combined set, respectively. The statistical results showed that the bruise ratio index was equivalent to the measured firmness but better than the predicted firmness in regard to effectiveness of bruise quantification, and the bruise ratio index had a strong correlation with human assessment (R2 = 0.78 - 0.83). Therefore, the proposed approach and the bruise ratio index are effective to non-destructively detect and quantify blueberry bruising.

Structural Characterization and Absolute Quantification of Microcystin Peptides Using Collision-Induced and Ultraviolet Photo-Dissociation Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Attard, Troy J.; Carter, Melissa D.; Fang, Mengxuan; Johnson, Rudolph C.; Reid, Gavin E.

2018-05-01

Microcystin (MC) peptides produced by cyanobacteria pose a hepatotoxic threat to human health upon ingestion from contaminated drinking water. While rapid MC identification and quantification in contaminated body fluids or tissue samples is important for patient treatment and outcomes, conventional immunoassay-based measurement strategies typically lack the specificity required for unambiguous determination of specific MC variants, whose toxicity can significantly vary depending on their structures. Furthermore, the unambiguous identification and accurate quantitation of MC variants using tandem mass spectrometry (MS/MS)-based methods can be limited due to a current lack of appropriate stable isotope-labeled internal standards. To address these limitations, we have systematically examined here the sequence and charge state dependence to the formation and absolute abundance of both "global" and "variant-specific" product ions from representative MC-LR, MC-YR, MC-RR, and MC-LA peptides, using higher-energy collisional dissociation (HCD)-MS/MS, ion-trap collision-induced dissociation (CID)-MS/MS and CID-MS3, and 193 nm ultraviolet photodissociation (UPVD)-MS/MS. HCD-MS/MS was found to provide the greatest detection sensitivity for both global and variant-specific product ions in each of the MC variants, except for MC-YR where a variant-specific product uniquely formed via UPVD-MS/MS was observed with the greatest absolute abundance. A simple methodology for the preparation and characterization of 18O-stable isotope-labeled MC reference materials for use as internal standards was also developed. Finally, we have demonstrated the applicability of the methods developed herein for absolute quantification of MC-LR present in human urine samples, using capillary scale liquid chromatography coupled with ultra-high resolution / accurate mass spectrometry and HCD-MS/MS.

Detection of beryllium in digested autopsy tissues by inductively coupled plasma mass spectrometry using a high matrix interface configuration.

PubMed

Larivière, Dominic; Tremblay, Mélodie; Durand-Jézéquel, Myriam; Tolmachev, Sergei

2012-04-01

This article describes a robust methodology using the combination of instrumental design (high matrix interface-HMI), sample dilution and internal standardization for the quantification of beryllium (Be) in various digested autopsy tissues using inductively coupled plasma mass spectrometry. The applicability of rhodium as a proper internal standard for Be was demonstrated in three types of biological matrices (i.e., femur, hair, lung tissues). Using HMI, it was possible to achieve instrumental detection limits and sensitivity of 0.6 ng L(-1) and 157 cps L ng(-1), respectively. Resilience to high salt matrices of the HMI setup was also highlighted using bone mimicking solution ([Ca(2+)] = 26 to 1,400 mg L(-1)), providing a 14-fold increase in tolerance and a 2.7-fold decrease in method detection limit compared to optimized experimental conditions obtained without the HMI configuration. Precision of the methodology to detect low levels of Be in autopsy samples was demonstrated using hair and blood certified reference materials. Be concentration ranging from 0.015 to 255 μg kg(-1) in autopsy samples obtained from the U.S. Transuranium and Uranium Registries were measured using the methodology presented.

Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring*

PubMed Central

Lawless, Craig; Holman, Stephen W.; Brownridge, Philip; Lanthaler, Karin; Harman, Victoria M.; Watkins, Rachel; Hammond, Dean E.; Miller, Rebecca L.; Sims, Paul F. G.; Grant, Christopher M.; Eyers, Claire E.; Beynon, Robert J.

2016-01-01

Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800 Saccharomyces cerevisiae proteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a “gold-standard” reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies. PMID:26750110

Determination of the neuropharmacological drug nodakenin in rat plasma and brain tissues by liquid chromatography tandem mass spectrometry: Application to pharmacokinetic studies.

PubMed

Song, Yingshi; Yan, Huiyu; Xu, Jingbo; Ma, Hongxi

2017-09-01

A rapid and sensitive liquid chromatography tandem mass spectrometry detection using selected reaction monitoring in positive ionization mode was developed and validated for the quantification of nodakenin in rat plasma and brain. Pareruptorin A was used as internal standard. A single step liquid-liquid extraction was used for plasma and brain sample preparation. The method was validated with respect to selectivity, precision, accuracy, linearity, limit of quantification, recovery, matrix effect and stability. Lower limit of quantification of nodakenin was 2.0 ng/mL in plasma and brain tissue homogenates. Linear calibration curves were obtained over concentration ranges of 2.0-1000 ng/mL in plasma and brain tissue homogenates for nodakenin. Intra-day and inter-day precisions (relative standard deviation, RSD) were <15% in both biological media. This assay was successfully applied to plasma and brain pharmacokinetic studies of nodakenin in rats after intravenous administration. Copyright © 2017 John Wiley & Sons, Ltd.

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

PubMed Central

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

PubMed

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the

Improved quantification for local regions of interest in preclinical PET imaging

NASA Astrophysics Data System (ADS)

Cal-González, J.; Moore, S. C.; Park, M.-A.; Herraiz, J. L.; Vaquero, J. J.; Desco, M.; Udias, J. M.

2015-09-01

In Positron Emission Tomography, there are several causes of quantitative inaccuracy, such as partial volume or spillover effects. The impact of these effects is greater when using radionuclides that have a large positron range, e.g. 68Ga and 124I, which have been increasingly used in the clinic. We have implemented and evaluated a local projection algorithm (LPA), originally evaluated for SPECT, to compensate for both partial-volume and spillover effects in PET. This method is based on the use of a high-resolution CT or MR image, co-registered with a PET image, which permits a high-resolution segmentation of a few tissues within a volume of interest (VOI) centered on a region within which tissue-activity values need to be estimated. The additional boundary information is used to obtain improved activity estimates for each tissue within the VOI, by solving a simple inversion problem. We implemented this algorithm for the preclinical Argus PET/CT scanner and assessed its performance using the radionuclides 18F, 68Ga and 124I. We also evaluated and compared the results obtained when it was applied during the iterative reconstruction, as well as after the reconstruction as a postprocessing procedure. In addition, we studied how LPA can help to reduce the ‘spillover contamination’, which causes inaccurate quantification of lesions in the immediate neighborhood of large, ‘hot’ sources. Quantification was significantly improved by using LPA, which provided more accurate ratios of lesion-to-background activity concentration for hot and cold regions. For 18F, the contrast was improved from 3.0 to 4.0 in hot lesions (when the true ratio was 4.0) and from 0.16 to 0.06 in cold lesions (true ratio  =  0.0), when using the LPA postprocessing. Furthermore, activity values estimated within the VOI using LPA during reconstruction were slightly more accurate than those obtained by post-processing, while also visually improving the image contrast and uniformity

Improved quantification for local regions of interest in preclinical PET imaging

PubMed Central

Cal-González, J.; Moore, S. C.; Park, M.-A.; Herraiz, J. L.; Vaquero, J. J.; Desco, M.; Udias, J. M.

2015-01-01

In Positron Emission Tomography, there are several causes of quantitative inaccuracy, such as partial volume or spillover effects. The impact of these effects is greater when using radionuclides that have a large positron range, e.g., 68Ga and 124I, which have been increasingly used in the clinic. We have implemented and evaluated a local projection algorithm (LPA), originally evaluated for SPECT, to compensate for both partial-volume and spillover effects in PET. This method is based on the use of a high-resolution CT or MR image, co-registered with a PET image, which permits a high-resolution segmentation of a few tissues within a volume of interest (VOI) centered on a region within which tissue-activity values need to be estimated. The additional boundary information is used to obtain improved activity estimates for each tissue within the VOI, by solving a simple inversion problem. We implemented this algorithm for the preclinical Argus PET/CT scanner and assessed its performance using the radionuclides 18F, 68Ga and 124I. We also evaluated and compared the results obtained when it was applied during the iterative reconstruction, as well as after the reconstruction as a postprocessing procedure. In addition, we studied how LPA can help to reduce the “spillover contamination”, which causes inaccurate quantification of lesions in the immediate neighborhood of large, “hot” sources. Quantification was significantly improved by using LPA, which provided more accurate ratios of lesion-to-background activity concentration for hot and cold regions. For 18F, the contrast was improved from 3.0 to 4.0 in hot lesions (when the true ratio was 4.0) and from 0.16 to 0.06 in cold lesions (true ratio = 0.0), when using the LPA postprocessing. Furthermore, activity values estimated within the VOI using LPA during reconstruction were slightly more accurate than those obtained by post-processing, while also visually improving the image contrast and uniformity within the VOI

Fingerprinting and quantification of GMOs in the agro-food sector.

PubMed

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

2003-01-01

Most strategies for analyzing GMOs in plants and derived food and feed products, are based on the polymerase chain reaction (PCR) technique. In conventional PCR methods, a 'known' sequence between two specific primers is amplified. To the contrary, with the 'anchor PCR' technique, unknown sequences adjacent to a known sequence, can be amplified. Because T-DNA/plant border sequences are being amplified, anchor PCR is the perfect tool for unique identification of transgenes, including non-authorized GMOs. In this work, anchor PCR was applied to characterize the 'transgene locus' and to clarify the complete molecular structure of at least six different commercial transgenic plants. Based on sequences of T-DNA/plant border junctions, obtained by anchor PCR, event specific primers were developed. The junction fragments, together with endogeneous reference gene targets, were cloned in plasmids. The latter were then used as event specific calibrators in real-time PCR, a new technique for the accurate relative quantification of GMOs. We demonstrate here the importance of anchor PCR for identification and the usefulness of plasmid DNA calibrators in quantification strategies for GMOs, throughout the agro-food sector.

Integrative analysis of 111 reference human epigenomes

PubMed Central

Kundaje, Anshul; Meuleman, Wouter; Ernst, Jason; Bilenky, Misha; Yen, Angela; Kheradpour, Pouya; Zhang, Zhizhuo; Heravi-Moussavi, Alireza; Liu, Yaping; Amin, Viren; Ziller, Michael J; Whitaker, John W; Schultz, Matthew D; Sandstrom, Richard S; Eaton, Matthew L; Wu, Yi-Chieh; Wang, Jianrong; Ward, Lucas D; Sarkar, Abhishek; Quon, Gerald; Pfenning, Andreas; Wang, Xinchen; Claussnitzer, Melina; Coarfa, Cristian; Harris, R Alan; Shoresh, Noam; Epstein, Charles B; Gjoneska, Elizabeta; Leung, Danny; Xie, Wei; Hawkins, R David; Lister, Ryan; Hong, Chibo; Gascard, Philippe; Mungall, Andrew J; Moore, Richard; Chuah, Eric; Tam, Angela; Canfield, Theresa K; Hansen, R Scott; Kaul, Rajinder; Sabo, Peter J; Bansal, Mukul S; Carles, Annaick; Dixon, Jesse R; Farh, Kai-How; Feizi, Soheil; Karlic, Rosa; Kim, Ah-Ram; Kulkarni, Ashwinikumar; Li, Daofeng; Lowdon, Rebecca; Mercer, Tim R; Neph, Shane J; Onuchic, Vitor; Polak, Paz; Rajagopal, Nisha; Ray, Pradipta; Sallari, Richard C; Siebenthall, Kyle T; Sinnott-Armstrong, Nicholas; Stevens, Michael; Thurman, Robert E; Wu, Jie; Zhang, Bo; Zhou, Xin; Beaudet, Arthur E; Boyer, Laurie A; De Jager, Philip; Farnham, Peggy J; Fisher, Susan J; Haussler, David; Jones, Steven; Li, Wei; Marra, Marco; McManus, Michael T; Sunyaev, Shamil; Thomson, James A; Tlsty, Thea D; Tsai, Li-Huei; Wang, Wei; Waterland, Robert A; Zhang, Michael; Chadwick, Lisa H; Bernstein, Bradley E; Costello, Joseph F; Ecker, Joseph R; Hirst, Martin; Meissner, Alexander; Milosavljevic, Aleksandar; Ren, Bing; Stamatoyannopoulos, John A; Wang, Ting; Kellis, Manolis

2015-01-01

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but a similar reference has lacked for epigenomic studies. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection to-date of human epigenomes for primary cells and tissues. Here, we describe the integrative analysis of 111 reference human epigenomes generated as part of the program, profiled for histone modification patterns, DNA accessibility, DNA methylation, and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically-relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation, and human disease. PMID:25693563

Integrative analysis of 111 reference human epigenomes.

PubMed

Kundaje, Anshul; Meuleman, Wouter; Ernst, Jason; Bilenky, Misha; Yen, Angela; Heravi-Moussavi, Alireza; Kheradpour, Pouya; Zhang, Zhizhuo; Wang, Jianrong; Ziller, Michael J; Amin, Viren; Whitaker, John W; Schultz, Matthew D; Ward, Lucas D; Sarkar, Abhishek; Quon, Gerald; Sandstrom, Richard S; Eaton, Matthew L; Wu, Yi-Chieh; Pfenning, Andreas R; Wang, Xinchen; Claussnitzer, Melina; Liu, Yaping; Coarfa, Cristian; Harris, R Alan; Shoresh, Noam; Epstein, Charles B; Gjoneska, Elizabeta; Leung, Danny; Xie, Wei; Hawkins, R David; Lister, Ryan; Hong, Chibo; Gascard, Philippe; Mungall, Andrew J; Moore, Richard; Chuah, Eric; Tam, Angela; Canfield, Theresa K; Hansen, R Scott; Kaul, Rajinder; Sabo, Peter J; Bansal, Mukul S; Carles, Annaick; Dixon, Jesse R; Farh, Kai-How; Feizi, Soheil; Karlic, Rosa; Kim, Ah-Ram; Kulkarni, Ashwinikumar; Li, Daofeng; Lowdon, Rebecca; Elliott, GiNell; Mercer, Tim R; Neph, Shane J; Onuchic, Vitor; Polak, Paz; Rajagopal, Nisha; Ray, Pradipta; Sallari, Richard C; Siebenthall, Kyle T; Sinnott-Armstrong, Nicholas A; Stevens, Michael; Thurman, Robert E; Wu, Jie; Zhang, Bo; Zhou, Xin; Beaudet, Arthur E; Boyer, Laurie A; De Jager, Philip L; Farnham, Peggy J; Fisher, Susan J; Haussler, David; Jones, Steven J M; Li, Wei; Marra, Marco A; McManus, Michael T; Sunyaev, Shamil; Thomson, James A; Tlsty, Thea D; Tsai, Li-Huei; Wang, Wei; Waterland, Robert A; Zhang, Michael Q; Chadwick, Lisa H; Bernstein, Bradley E; Costello, Joseph F; Ecker, Joseph R; Hirst, Martin; Meissner, Alexander; Milosavljevic, Aleksandar; Ren, Bing; Stamatoyannopoulos, John A; Wang, Ting; Kellis, Manolis

2015-02-19

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Multimodal noninvasive monitoring of soft tissue wound healing.

PubMed

Bodo, Michael; Settle, Timothy; Royal, Joseph; Lombardini, Eric; Sawyer, Evelyn; Rothwell, Stephen W

2013-12-01

Here we report results of non-invasive measurements of indirect markers of soft tissue healing of traumatic wounds in an observational swine study and describe the quantification of analog physiological signals. The primary purpose of the study was to measure bone healing of fractures with four different wound treatments. A second purpose was to quantify soft tissue wound healing by measuring the following indirect markers: (1) tissue oxygenation, (2) fluid content, and (3) blood flow, which were all measured by non-invasive modalities, measured with available devices. Tissue oxygenation was measured by near infrared spectroscopy; fluid content was measured by bipolar bio-impedance; and blood flow was measured by Doppler ultrasound. Immediately after comminuted femur fractures were produced in the right hind legs of thirty anesthetized female Yorkshire swine, one of four wound treatments was instilled into each wound. The four wound treatments were as follows: salmon fibrinogen/thrombin-n = 8; commercial bone filler matrix-n = 7; bovine collagen-n = 8; porcine fibrinogen/thrombin-n = 7. Fractures were stabilized with an external fixation device. Immediately following wound treatments, measurements were made of tissue oxygenation, fluid content and blood flow; these measurements were repeated weekly for 3 weeks after surgery. Analog signals of each modality were recorded on both the wounded (right) hind leg and the healthy (left) hind leg, for comparison purposes. Data were processed off-line. The mean values of 10-s periods were calculated for right-left leg comparison. ANOVA was applied for statistical analysis. Results of the bone healing studies are published separately (Rothwell et al. in J Spec Oper Med 13:7-18, 2013). For soft tissue wounds, healing did not differ significantly among the four wound treatments; however, regional oxygenation of wounds treated with salmon fibrinogen/thrombin showed slightly different time trends. Further studies are

Seasonal changes of nucleotides in mussel (Mytilus galloprovincialis) mantle tissue.

PubMed

Blanco, S L; Suárez, M P; San Juan, F

2006-03-01

Seasonal variations of nucleotides in Mytilus galloprovincialis mantle tissue were analyzed. Separation and quantification was achieved by reversed-phase high-performance liquid chromatography. Total nucleotides show a pronounced seasonal variation with maximum and minimum values in autumn and spring, respectively. Adenine nucleotides accounted for the major part in spring and summer, guanosine and cytidine nucleotides in winter; uridine nucleotides were relatively constant throughout the year. Their inverse variation suggests inter-conversion among them and the maintenance of the potential cell energy in winter by other triphosphate nucleotides different from ATP. These results reflect environmental and nutritional conditions, and also the reserves and gametogenic cycles taking place in M. galloprovincialis mantle tissue.

Application of a liquid chromatography-tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion studies of Dactylicapnos scandens in rats.

PubMed

Guo, Changchuan; Jiang, Yan; Li, Li; Hong, Lan; Wang, Yuqing; Shen, Qian; Lou, Yan; Hu, Haihong; Zhou, Hui; Yu, Lushan; Jiang, Huidi; Zeng, Su

2013-02-23

The herbal ingredients of isocorydine and protopine were isolated from Dactylicapnos scandens. This study was aimed at developing a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to quantify isocorydine and protopine in rat plasma and tissues for pharmacokinetic, tissue distribution and excretion studies. Biological samples were processed with ethyl acetate extraction, and corydaline was chosen as the internal standard (IS). The analytes were separated by a C(18) column and detected with a triple quadrupole mass spectrometer using positive ion ESI in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 342.0→278.9 for isocorydine, 354.1→188.0 for protopine and 370.0→192.0 for IS, respectively. Excellent linearity was observed over the concentration range between 10 and 3000 ng/mL for isocorydine and 10-300 ng/mL for protopine. The lower limit of quantification (LLOQ) was 10 ng/mL for both isocorydine and protopine. This novel method was rapid, accurate, high sensitive and high selective. It was successfully applied to the pharmacokinetic, tissue distribution and excretion studies of D. scandens. These preclinical data of D. scandens would be useful for the clinical reference. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantification of fatal helium exposure following self-administration.

PubMed

Malbranque, S; Mauillon, D; Turcant, A; Rouge-Maillart, C; Mangin, P; Varlet, V

2016-11-01

Helium is nontoxic at standard conditions, plays no biological role, and is found in trace amounts in human blood. Helium can be dangerous if inhaled to excess, since it is a simple tissue hypoxia and so displaces the oxygen needed for normal respiration. This report presents a fatal case of a middle-aged male victim who died from self-administered helium exposure. For the first time, the quantification of the helium levels in gastric and lung air and in blood samples was achieved using gas chromatography-mass spectrometry after airtight sampling. The results of the toxicological investigation showed that death was caused directly by helium exposure. However, based on the pathomorphological changes detected during the forensic autopsy, we suppose that the fatal outcome was the result of the lack of oxygen after inhalation.

[Effect of elastic strain rate ratio method and virtual touch tissue quantification on the diagnosis of breast masses].

PubMed

Gong, LiJie; He, Yan; Tian, Peng; Yan, Yan

2016-07-01

To determine the effect of elastic strain rate ratio method and virtual touch tissue quantification (VTQ) on the diagnosis of breast masses.
 Sixty female patients with breast cancer, who received surgical treatment in Daqing Oilfield General Hospital, were enrolled. All patients signed the informed consent paperwork and they were treated by routine ultrasound examination, compression elastography (CE) examination, and VTQ examination in turn. Strain ratio (SR) was checked by CE and shear wave velocity (SWV) value was measured by VTQ. The diagnostic values of different methods were evaluated by receiver operating characteristic (ROC) curves in the diagnosis of benign and malignant breast tumors.
 The maximum diameter and SWV value of the benign tumors were lower than those of the malignant tumors, and the SR ratio of benign masses was higher than that of malignant tumors (P<0.01). The AUC, sensitivity and specificity for elastic strain rate and VTQ for single or combined use were higher than those of conventional ultrasound (0.904, 97.5%, 69.2%; 0.946, 87.5%, 87.2%; 0.976, 90%, 97.4% vs 0.783, 85%, 61.5%). The AUC and specificity of VTQ were higher than those of the elastic strain rate (0.946, 87.2% vs 0.904, 69.2%), but the sensitivity of VTQ was higher than that of the latter (87.5% vs 97.5%). The AUC and specificity for combination of both methods were higher than those of single method, but the sensitivity was lower than that of the elastic strain rate. 
 Combination of elastic strain rate ratio method with VTQ possesses the best diagnostic value and the highest diagnostic accuracy in the diagnosis of breast mass than that used alone.

Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins.

PubMed

Mueller, Jenna L; Harmany, Zachary T; Mito, Jeffrey K; Kennedy, Stephanie A; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G; Willett, Rebecca M; Brown, J Quincy; Ramanujam, Nimmi

2013-01-01

To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. TISSUE EXCISED FROM A GENETICALLY ENGINEERED MOUSE MODEL OF SARCOMA WAS IMAGED USING A SUBCELLULAR RESOLUTION MICROENDOSCOPE AFTER TOPICAL APPLICATION OF A FLUORESCENT ANATOMICAL CONTRAST AGENT: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator.

PubMed

Rossano, Adam J; Romero, Michael F

2017-08-11

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H + ) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via

Quantitative bioimaging of p-boronophenylalanine in thin liver tissue sections as a tool for treatment planning in boron neutron capture therapy.

PubMed

Reifschneider, Olga; Schütz, Christian L; Brochhausen, Christoph; Hampel, Gabriele; Ross, Tobias; Sperling, Michael; Karst, Uwe

2015-03-01

An analytical method using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was developed and applied to assess enrichment of 10B-containing p-boronophenylalanine-fructose (BPA-f) and its pharmacokinetic distribution in human tissues after application for boron neutron capture therapy (BNCT). High spatial resolution (50 μm) and limits of detection in the low parts-per-billion range were achieved using a Nd:YAG laser of 213 nm wavelength. External calibration by means of 10B-enriched standards based on whole blood proved to yield precise quantification results. Using this calibration method, quantification of 10B in cancerous and healthy tissue was carried out. Additionally, the distribution of 11B was investigated, providing 10B enrichment in the investigated tissues. Quantitative imaging of 10B by means of LA-ICP-MS was demonstrated as a new option to characterise the efficacy of boron compounds for BNCT.

Improved selenium recovery from tissue with modified sample decomposition

USGS Publications Warehouse

Brumbaugh, W. G.; Walther, M.J.

1991-01-01

The present paper describes a simple modification of a recently reported decomposition method for determination of selenium in biological tissue by hydride generation atomic absorption. The modified method yielded slightly higher selenium recoveries (3-4%) for selected reference tissues and fish tissue spiked with selenomethionine. Radiotracer experiments indicated that the addition of a small volume of hydrochloric acid to the wet digestate mixture reduced slight losses of selenium as the sample initially went to dryness before ashing. With the modified method, selenium spiked as selenomethionine behaved more like the selenium in reference tissues than did the inorganic spike forms when this digestion modification was used.

Calibration-free quantification of absolute oxygen saturation based on the dynamics of photoacoustic signals

PubMed Central

Xia, Jun; Danielli, Amos; Liu, Yan; Wang, Lidai; Maslov, Konstantin; Wang, Lihong V.

2014-01-01

Photoacoustic tomography (PAT) is a hybrid imaging technique that has broad preclinical and clinical applications. Based on the photoacoustic effect, PAT directly measures specific optical absorption, which is the product of the tissue-intrinsic optical absorption coefficient and the local optical fluence. Therefore, quantitative PAT, such as absolute oxygen saturation (sO2) quantification, requires knowledge of the local optical fluence, which can be estimated only through invasive measurements or sophisticated modeling of light transportation. In this work, we circumvent this requirement by taking advantage of the dynamics in sO2. The new method works when the sO2 transition can be simultaneously monitored with multiple wavelengths. For each wavelength, the ratio of photoacoustic amplitudes measured at different sO2 states is utilized. Using the ratio cancels the contribution from optical fluence and allows calibration-free quantification of absolute sO2. The new method was validated through both phantom and in vivo experiments. PMID:23903146

Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.

PubMed

Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

2014-05-19

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3--high copy number group, TST-1 and PRR-1--medium copy number group, P4H-1, APRT-2 and CYC-2--low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

Certification of the methylmercury content in SRM 2977 mussel tissue (organic contaminants and trace elements) and SRM 1566b oyster tissue.

PubMed

Tutschku, S; Schantz, M M; Horvat, M; Logar, M; Akagi, H; Emons, H; Levenson, M; Wise, S A

2001-02-01

The methylmercury content in two new marine bivalve mollusk tissue Standard Reference Materials (SRMs) has been certified using results of analyses from the National Institute of Standards and Technology (NIST) and two other laboratories. The certified concentrations of methylmercury were established based on the results from four and six different (independent) analytical methods, respectively, for SRM 1566b Oyster Tissue (13.2 +/- 0.7 microg/kg) and SRM 2977 Mussel Tissue (organic contaminants and trace elements) (36.2 +/- 1.7 microg/kg). The certified concentration of methylmercury in SRM 1566b is among the lowest in any certified reference material (CRM).

Thyroid tissue constituents characterization and application to in vivo studies by broadband (600-1200 nm) diffuse optical spectroscopy

NASA Astrophysics Data System (ADS)

Konugolu Venkata Sekar, Sanathana; Farina, Andrea; dalla Mora, Alberto; Taroni, Paola; Lindner, Claus; Mora, Mireia; Farzam, Parisa; Pagliazzi, Marco; Squarcia, Mattia; Halperin, Irene; Hanzu, Felicia A.; Dehghani, Hamid; Durduran, Turgut; Pifferi, Antonio

2017-07-01

We present the first broadband (600-1100 nm) diffuse optical characterization of thyroglobulin and tyrosine, which are thyroid-specific tissue constituents. In-vivo measurements at the thyroid region enabled their quantification for functional and diagnostic applications.

Diffuse optical microscopy for quantification of depth-dependent epithelial backscattering in the cervix

NASA Astrophysics Data System (ADS)

Bodenschatz, Nico; Lam, Sylvia; Carraro, Anita; Korbelik, Jagoda; Miller, Dianne M.; McAlpine, Jessica N.; Lee, Marette; Kienle, Alwin; MacAulay, Calum

2016-06-01

A fiber optic imaging approach is presented using structured illumination for quantification of almost pure epithelial backscattering. We employ multiple spatially modulated projection patterns and camera-based reflectance capture to image depth-dependent epithelial scattering. The potential diagnostic value of our approach is investigated on cervical ex vivo tissue specimens. Our study indicates a strong backscattering increase in the upper part of the cervical epithelium caused by dysplastic microstructural changes. Quantization of relative depth-dependent backscattering is confirmed as a potentially useful diagnostic feature for detection of precancerous lesions in cervical squamous epithelium.

Proteomic evaluation of human umbilical cord tissue exposed to polybrominated diphenyl ethers in an e-waste recycling area.

PubMed

Li, Minghui; Huo, Xia; Pan, Yukui; Cai, Haoxing; Dai, Yifeng; Xu, Xijin

2018-02-01

Parental exposure to polybrominated diphenyl ethers (PBDEs) is associated with adverse birth outcomes. This study aims to examine differentially-expressed protein profiles in umbilical cord tissue, derived from mothers exposed to PBDEs, and investigate candidate biomarkers to reveal the underlying molecular mechanisms. Umbilical cord samples were obtained from women residing in an electronic waste (e-waste) recycling area (Guiyu) and reference area (Haojiang) in China. The concentration of PBDEs in umbilical cord tissue was determined by gas chromatography and mass spectrometry (GC/MS). Isobaric tagging for relative and absolute quantification (iTRAQ)-based proteomic technology was conducted to analyze differentially-expressed protein profiles. The total PBDE concentration was approximately five-fold higher in umbilical cords from Guiyu than from Haojiang (median 71.92ng/g vs. 15.52ng/g lipid, P<0.01). Neonatal head circumference, body-mass index (BMI) and Apgar1 score were lower in Guiyu and negatively correlated with PBDE concentration (P<0.01). Proteomic analysis showed 697 proteins were differentially expressed in the e-waste-exposed group compared with the reference group. The differentially-expressed proteins were principally involved in antioxidant defense, apoptosis, cell structure and metabolism. Among them, catalase and glutathione S-transferase omega-1, were down-regulated, and cytochrome c was found to be up-regulated, changes which were further verified by enzyme-linked immunosorbent assays. These results suggest that an antioxidant imbalance and cell apoptosis in the umbilical cord following PBDE exposure is associated with neonatal birth outcomes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression stability and selection of optimal reference genes for gene expression normalization in early life stage rainbow trout exposed to cadmium and copper.

PubMed

Shekh, Kamran; Tang, Song; Niyogi, Som; Hecker, Markus

2017-09-01

Gene expression analysis represents a powerful approach to characterize the specific mechanisms by which contaminants interact with organisms. One of the key considerations when conducting gene expression analyses using quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the selection of appropriate reference genes, which is often overlooked. Specifically, to reach meaningful conclusions when using relative quantification approaches, expression levels of reference genes must be highly stable and cannot vary as a function of experimental conditions. However, to date, information on the stability of commonly used reference genes across developmental stages, tissues and after exposure to contaminants such as metals is lacking for many vertebrate species including teleost fish. Therefore, in this study, we assessed the stability of expression of 8 reference gene candidates in the gills and skin of three different early life-stages of rainbow trout after acute exposure (24h) to two metals, cadmium (Cd) and copper (Cu) using qPCR. Candidate housekeeping genes were: beta actin (b-actin), DNA directed RNA polymerase II subunit I (DRP2), elongation factor-1 alpha (EF1a), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PD), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein L8 (RPL8), and 18S ribosomal RNA (18S). Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative ΔCt method were employed to systematically evaluate the expression stability of these candidate genes under control and exposed conditions as well as across three different life-stages. Finally, stability of genes was ranked by taking geometric means of the ranks established by the different methods. Stability of reference genes was ranked in the following order (from lower to higher stability): HPRT

Method for accurate quantitation of background tissue optical properties in the presence of emission from a strong fluorescence marker

NASA Astrophysics Data System (ADS)

Bravo, Jaime; Davis, Scott C.; Roberts, David W.; Paulsen, Keith D.; Kanick, Stephen C.

2015-03-01

Quantification of targeted fluorescence markers during neurosurgery has the potential to improve and standardize surgical distinction between normal and cancerous tissues. However, quantitative analysis of marker fluorescence is complicated by tissue background absorption and scattering properties. Correction algorithms that transform raw fluorescence intensity into quantitative units, independent of absorption and scattering, require a paired measurement of localized white light reflectance to provide estimates of the optical properties. This study focuses on the unique problem of developing a spectral analysis algorithm to extract tissue absorption and scattering properties from white light spectra that contain contributions from both elastically scattered photons and fluorescence emission from a strong fluorophore (i.e. fluorescein). A fiber-optic reflectance device was used to perform measurements in a small set of optical phantoms, constructed with Intralipid (1% lipid), whole blood (1% volume fraction) and fluorescein (0.16-10 μg/mL). Results show that the novel spectral analysis algorithm yields accurate estimates of tissue parameters independent of fluorescein concentration, with relative errors of blood volume fraction, blood oxygenation fraction (BOF), and the reduced scattering coefficient (at 521 nm) of <7%, <1%, and <22%, respectively. These data represent a first step towards quantification of fluorescein in tissue in vivo.

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

PubMed

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

2017-04-01

Levo-tetrahydropalmatine (l-THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l-THP and its desmethyl metabolites l-corydalmine (l-CD) and l-corypalmine (l-CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid-liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed-phase Symmetry® C 18 column (4.6 × 150 mm, 5 μm) at 25°C. The mobile phase consisted of acetonitrile-methanol-10 mm ammonium phosphate (pH 3) (10:30:60, v/v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1-10,000 ng/mL. The intra- and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l-THP in rats. Taken together, the developed method can be applied for bioanalysis of l-THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples. Copyright © 2016 John Wiley & Sons, Ltd.

Practical considerations of image analysis and quantification of signal transduction IHC staining.

PubMed

Grunkin, Michael; Raundahl, Jakob; Foged, Niels T

2011-01-01

The dramatic increase in computer processing power in combination with the availability of high-quality digital cameras during the last 10 years has fertilized the grounds for quantitative microscopy based on digital image analysis. With the present introduction of robust scanners for whole slide imaging in both research and routine, the benefits of automation and objectivity in the analysis of tissue sections will be even more obvious. For in situ studies of signal transduction, the combination of tissue microarrays, immunohistochemistry, digital imaging, and quantitative image analysis will be central operations. However, immunohistochemistry is a multistep procedure including a lot of technical pitfalls leading to intra- and interlaboratory variability of its outcome. The resulting variations in staining intensity and disruption of original morphology are an extra challenge for the image analysis software, which therefore preferably should be dedicated to the detection and quantification of histomorphometrical end points.

MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions

NASA Astrophysics Data System (ADS)

Shipunova, V. O.; Nikitin, M. P.; Nikitin, P. I.; Deyev, S. M.

2016-06-01

Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method

Photoacoustic physio-chemical analysis and its implementation in deep tissue with a catheter setup

NASA Astrophysics Data System (ADS)

Xu, Guan; Meng, Zhou-xian; Lin, Jian-die D.; Cheng, Qian; Wang, Xueding

2015-03-01

Photoacoustic (PA) measurements encode the information associated with both physical microstructures and chemical contents in biological tissues. A two-dimensional physio-chemical spectrogram (PCS) can be formulated by combining the power spectra of PA signals acquired at a series of optical wavelengths. The analysis of PCS, or namely PA physio-chemical analysis (PAPCA), enables the quantification of the relative concentrations and the spatial distributions of a variety of chemical components in the tissue. This study validated the feasibility of PAPCA in characterizing liver conditions during the progression of non-alcoholic fatty liver disease. A catheter based setup facilitating measurement in deep tissues was also tested.

Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

PubMed

Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2013-10-10

Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots. Copyright © 2013 Elsevier B.V. All rights reserved.

Processing and domain selection: Quantificational variability effects

PubMed Central

Harris, Jesse A.; Clifton, Charles; Frazier, Lyn

2014-01-01

Three studies investigated how readers interpret sentences with variable quantificational domains, e.g., The army was mostly in the capital, where mostly may quantify over individuals or parts (Most of the army was in the capital) or over times (The army was in the capital most of the time). It is proposed that a general conceptual economy principle, No Extra Times (Majewski 2006, in preparation), discourages the postulation of potentially unnecessary times, and thus favors the interpretation quantifying over parts. Disambiguating an ambiguously quantified sentence to a quantification over times interpretation was rated as less natural than disambiguating it to a quantification over parts interpretation (Experiment 1). In an interpretation questionnaire, sentences with similar quantificational variability were constructed so that both interpretations of the sentence would require postulating multiple times; this resulted in the elimination of the preference for a quantification over parts interpretation, suggesting the parts preference observed in Experiment 1 is not reducible to a lexical bias of the adverb mostly (Experiment 2). An eye movement recording study showed that, in the absence of prior evidence for multiple times, readers exhibit greater difficulty when reading material that forces a quantification over times interpretation than when reading material that allows a quantification over parts interpretation (Experiment 3). These experiments contribute to understanding readers’ default assumptions about the temporal properties of sentences, which is essential for understanding the selection of a domain for adverbial quantifiers and, more generally, for understanding how situational constraints influence sentence processing. PMID:25328262

Concise Review: Mind the Gap: Challenges in Characterizing and Quantifying Cell- and Tissue-Based Therapies for Clinical Translation

PubMed Central

Rayment, Erin A; Williams, David J

2010-01-01

There are many challenges associated with characterizing and quantifying cells for use in cell- and tissue-based therapies. From a regulatory perspective, these advanced treatments must not only be safe and effective but also be made by high-quality manufacturing processes that allow for on-time delivery of viable products. Although sterility assays can be adapted from conventional bioprocessing, cell- and tissue-based therapies require more stringent safety assessments, especially in relation to use of animal products, immune reaction, and potential instability due to extended culture times. Furthermore, cell manufacturers who plan to use human embryonic stem cells in their therapies need to be particularly stringent in their final purification steps, due to the unrestricted growth potential of these cells. This review summarizes the current issues in characterization and quantification for cell- and tissue-based therapies, dividing these challenges into the regulatory themes of safety, potency, and manufacturing quality. It outlines current assays in use, as well as highlights the limits of many of these product release tests. Mode of action is discussed, with particular reference to in vitro surrogate assays that can be used to provide information to correlate with proposed in vivo patient efficacy. Importantly, this review highlights the requirement for basic research to improve current knowledge on the in vivo fate of these treatments; as well as an improved stakeholder negotiation process to identify the measurement requirements that will ensure the manufacture of the best possible cell- and tissue-based therapies within the shortest timeframe for the most patient benefit. PMID:20333747

Validation of a Sulfuric Acid Digestion Method for Inductively Coupled Plasma Mass Spectrometry Quantification of TiO2 Nanoparticles.

PubMed

Watkins, Preston S; Castellon, Benjamin T; Tseng, Chiyen; Wright, Moncie V; Matson, Cole W; Cobb, George P

2018-04-13

A consistent analytical method incorporating sulfuric acid (H 2 SO 4 ) digestion and ICP-MS quantification has been developed for TiO 2 quantification in biotic and abiotic environmentally relevant matrices. Sample digestion in H 2 SO 4 at 110°C provided consistent results without using hydrofluoric acid or microwave digestion. Analysis of seven replicate samples for four matrices on each of 3 days produced Ti recoveries of 97% ± 2.5%, 91 % ± 4.0%, 94% ± 1.8%, and 73 % ± 2.6% (mean ± standard deviation) from water, fish tissue, periphyton, and sediment, respectively. The method demonstrated consistent performance in analysis of water collected over a 1 month.

Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks

PubMed Central

2010-01-01

Background Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum). Results Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals with single hypoxic insult and 10 individuals with hypoxia preconditioning (8 hypoxic insults, 12 hours apart). We produced sequence information for reference gene candidates and monitored mRNA expression levels in four tissues: cerebellum, heart, gill and eye. The stability of the genes was examined with analysis of variance, geNorm and NormFinder. The best ranking genes in our study were eukaryotic translation elongation factor 1 beta (eef1b), ubiquitin (ubq) and polymerase (RNA) II (DNA directed) polypeptide F (polr2f). The performance of the ribosomal protein L6 (rpl6) was tissue-dependent. Notably, in one tissue the analysis of variance indicated statistically significant differences between treatments for genes that were ranked as the most stable candidates by reference gene software. Conclusions Our results indicate that eef1b and ubq are generally the most suitable reference genes for the conditions and tissues in the present epaulette shark studies. These genes could also be potential reference gene candidates for other physiological studies examining stress in elasmobranchs. The results emphasise the importance of inter-group variation in reference gene evaluation. PMID:20416043

Origin of the subepidermal tissue in Piper L. leaves.

PubMed

Nakamura, A T; Simão, E; Silva, L; Torres, G A

2015-05-01

Studies on the anatomy of Piper leaves demonstrate the presence of a subepidermal tissue distinct from the adjacent epidermis, which cells show thin walls and hyaline contents. Some authors consider such cells a hypodermal tissue, while others refer to them as components of a multiple epidermis. In this study, the nature of this subepidermal tissue was investigated through the analysis of leaf ontogeny in three Piper species. The analysis showed that the referred tissue originates from the ground meristem and, thus, should be considered a hypodermis. The studied species suggests that the role of the hypodermis would be to protect the photosynthetic apparatus from excess light, regulating the intensity of light reaching the chlorophyll parenchyma.

Quantification of genetically modified soya using strong anion exchange chromatography and time-of-flight mass spectrometry.

PubMed

Chang, Po-Chih; Reddy, P Muralidhar; Ho, Yen-Peng

2014-09-01

Stable-isotope dimethyl labeling was applied to the quantification of genetically modified (GM) soya. The herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) was labeled using a dimethyl labeling reagent, formaldehyde-H2 or -D2. The identification and quantification of CP4 EPSPS was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The CP4 EPSPS protein was separated from high abundance proteins using strong anion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the tryptic peptides from the samples and reference were labeled with formaldehyde-H2 and formaldehyde-D2, respectively. The two labeled pools were mixed and analyzed using MALDI-MS. The data showed a good correlation between the peak ratio of the H- and D-labeled peptides and the GM soya percentages at 0.5, 1, 3, and 5 %, with R (2) of 0.99. The labeling reagents are readily available. The labeling experiments and the detection procedures are simple. The approach is useful for the quantification of GM soya at a level as low as 0.5 %.

Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

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

Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora

Global proteomics approaches allow characterization of whole tissue lysates to an impressive depth. However, it is now increasingly recognized that to better understand the complexity of multicellular organisms, global protein profiling of specific spatially defined regions/substructures of tissues (i.e. spatially-resolved proteomics) is essential. Laser capture microdissection (LCM) enables microscopic isolation of defined regions of tissues preserving crucial spatial information. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, and that impact measurement robustness, quantification, and throughput. Here, we coupled LCM with a fully automated sample preparation workflow thatmore » with a single manual step allows: protein extraction, tryptic digestion, peptide cleanup and LC-MS/MS analysis of proteomes from microdissected tissues. Benchmarking against the current state of the art in ultrasensitive global proteomic analysis, our approach demonstrated significant improvements in quantification and throughput. Using our LCM-SNaPP proteomics approach, we characterized to a depth of more than 3,400 proteins, the ontogeny of protein changes during normal lung development in laser capture microdissected alveolar tissue containing ~4,000 cells per sample. Importantly, the data revealed quantitative changes for 350 low abundance transcription factors and signaling molecules, confirming earlier transcript-level observations and defining seven modules of coordinated transcription factor/signaling molecule expression patterns, suggesting that a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. Our LCM-proteomics approach facilitates efficient, spatially-resolved, ultrasensitive global proteomics analyses in high-throughput that will be enabling for several

Nondestructive Detection and Quantification of Blueberry Bruising using Near-infrared (NIR) Hyperspectral Reflectance Imaging

PubMed Central

Jiang, Yu; Li, Changying; Takeda, Fumiomi

2016-01-01

Currently, blueberry bruising is evaluated by either human visual/tactile inspection or firmness measurement instruments. These methods are destructive, time-consuming, and subjective. The goal of this paper was to develop a non-destructive approach for blueberry bruising detection and quantification. Experiments were conducted on 300 samples of southern highbush blueberry (Camellia, Rebel, and Star) and on 1500 samples of northern highbush blueberry (Bluecrop, Jersey, and Liberty) for hyperspectral imaging analysis, firmness measurement, and human evaluation. An algorithm was developed to automatically calculate a bruise ratio index (ratio of bruised to whole fruit area) for bruise quantification. The spectra of bruised and healthy tissues were statistically separated and the separation was independent of cultivars. Support vector machine (SVM) classification of the spectra from the regions of interest (ROIs) achieved over 94%, 92%, and 96% accuracy on the training set, independent testing set, and combined set, respectively. The statistical results showed that the bruise ratio index was equivalent to the measured firmness but better than the predicted firmness in regard to effectiveness of bruise quantification, and the bruise ratio index had a strong correlation with human assessment (R2 = 0.78 − 0.83). Therefore, the proposed approach and the bruise ratio index are effective to non-destructively detect and quantify blueberry bruising. PMID:27767050

Development of a real-time PCR method for the differential detection and quantification of four solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum).

PubMed

Chaouachi, Maher; El Malki, Redouane; Berard, Aurélie; Romaniuk, Marcel; Laval, Valérie; Brunel, Dominique; Bertheau, Yves

2008-03-26

The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.

With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

PubMed

Chapman, Joanne R; Waldenström, Jonas

2015-01-01

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

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

PubMed

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

2014-08-01

The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland. Copyright © 2014 the American Physiological Society.

Proteomic analysis of formalin-fixed paraffin-embedded renal tissue samples by label-free MS: assessment of overall technical variability and the impact of block age.

PubMed

Craven, Rachel A; Cairns, David A; Zougman, Alexandre; Harnden, Patricia; Selby, Peter J; Banks, Rosamonde E

2013-04-01

Protein profiling of formalin-fixed paraffin-embedded (FFPE) tissues has enormous potential for the discovery and validation of disease biomarkers. The aim of this study was to systematically characterize the effect of length of time of storage of such tissue blocks in pathology archives on the quality of data produced using label-free MS. Normal kidney and clear cell renal cell carcinoma tissues routinely collected up to 10 years prior to analysis were profiled using LC-MS/MS and the data analyzed using MaxQuant. Protein identities and quantification data were analyzed to examine differences between tissue blocks of different ages and assess the impact of technical and biological variability. An average of over 2000 proteins was seen in each sample with good reproducibility in terms of proteins identified and quantification for normal kidney tissue, with no significant effect of block age. Greater biological variability was apparent in the renal cell carcinoma tissue, possibly reflecting disease heterogeneity, but again there was good correlation between technical replicates and no significant effect of block age. These results indicate that archival storage time does not have a detrimental effect on protein profiling of FFPE tissues, supporting the use of such tissues in biomarker discovery studies. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling

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

Bager, C.L., E-mail: cba@nordicbioscience.com; Technical University of Denmark; Gudmann, N.

Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art toolsmore » to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFβ and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex

The importance of reference materials in doping-control analysis.

PubMed

Mackay, Lindsey G; Kazlauskas, Rymantas

2011-08-01

Currently a large range of pure substance reference materials are available for calibration of doping-control methods. These materials enable traceability to the International System of Units (SI) for the results generated by World Anti-Doping Agency (WADA)-accredited laboratories. Only a small number of prohibited substances have threshold limits for which quantification is highly important. For these analytes only the highest quality reference materials that are available should be used. Many prohibited substances have no threshold limits and reference materials provide essential identity confirmation. For these reference materials the correct identity is critical and the methods used to assess identity in these cases should be critically evaluated. There is still a lack of certified matrix reference materials to support many aspects of doping analysis. However, in key areas a range of urine matrix materials have been produced for substances with threshold limits, for example 19-norandrosterone and testosterone/epitestosterone (T/E) ratio. These matrix-certified reference materials (CRMs) are an excellent independent means of checking method recovery and bias and will typically be used in method validation and then regularly as quality-control checks. They can be particularly important in the analysis of samples close to threshold limits, in which measurement accuracy becomes critical. Some reference materials for isotope ratio mass spectrometry (IRMS) analysis are available and a matrix material certified for steroid delta values is currently under production. In other new areas, for example the Athlete Biological Passport, peptide hormone testing, designer steroids, and gene doping, reference material needs still need to be thoroughly assessed and prioritised.

Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins

PubMed Central

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-01-01

Purpose To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. Materials and Methods Tissue excised from a genetically engineered mouse model of sarcoma was imaged using a subcellular resolution microendoscope after topical application of a fluorescent anatomical contrast agent: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Results Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. Conclusion The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue. PMID:23824589

Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer*

PubMed Central

Gallien, Sebastien; Duriez, Elodie; Crone, Catharina; Kellmann, Markus; Moehring, Thomas; Domon, Bruno

2012-01-01

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein

HPC Analytics Support. Requirements for Uncertainty Quantification Benchmarks

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

Paulson, Patrick R.; Purohit, Sumit; Rodriguez, Luke R.

2015-05-01

This report outlines techniques for extending benchmark generation products so they support uncertainty quantification by benchmarked systems. We describe how uncertainty quantification requirements can be presented to candidate analytical tools supporting SPARQL. We describe benchmark data sets for evaluating uncertainty quantification, as well as an approach for using our benchmark generator to produce data sets for generating benchmark data sets.

Flow cytometry for receptor analysis from ex-vivo brain tissue in adult rat.

PubMed

Benoit, A; Guillamin, M; Aitken, P; Smith, P F; Philoxene, B; Sola, B; Poulain, L; Coquerel, A; Besnard, S

2018-07-01

Flow cytometry allows single-cell analysis of peripheral biological samples and is useful in many fields of research and clinical applications, mainly in hematology, immunology, and oncology. In the neurosciences, the flow cytometry separation method was first applied to stem cell extraction from healthy or cerebral tumour tissue and was more recently tested in order to phenotype brain cells, hippocampal neurogenesis, and to detect prion proteins. However, it remains sparsely applied in quantifying membrane receptors in relation to synaptic plasticity. We aimed to optimize a flow cytometric procedure for receptor quantification in neurons and non-neurons. A neural dissociation process, myelin separation, fixation, and membrane permeability procedures were optimized to maximize cell survival and analysis in hippocampal tissue obtained from adult rodents. We then aimed to quantify membrane muscarinic acetylcholine receptors (mAChRs) in rats with and without bilateral vestibular loss (BVL). mAChR's were quantified for neuronal and non-neuronal cells in the hippocampus and striatum following BVL. At day 30 but not at day 7 following BVL, there was a significant increase (P ≤ 0.05) in the percentage of neurons expressing M 2/4 mAChRs in both the hippocampus and the striatum. Here, we showed that flow cytometry appears to be a reliable method of membrane receptor quantification in ex-vivo brain tissue. Copyright © 2018 Elsevier B.V. All rights reserved.

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

USGS Publications Warehouse

Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

2017-01-01

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

Relative quantification in seed GMO analysis: state of art and bottlenecks.

PubMed

Chaouachi, Maher; Bérard, Aurélie; Saïd, Khaled

2013-06-01

Reliable quantitative methods are needed to comply with current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) and GMO-derived food and feed products with a minimum GMO content of 0.9 %. The implementation of EU Commission Recommendation 2004/787/EC on technical guidance for sampling and detection which meant as a helpful tool for the practical implementation of EC Regulation 1830/2003, which states that "the results of quantitative analysis should be expressed as the number of target DNA sequences per target taxon specific sequences calculated in terms of haploid genomes". This has led to an intense debate on the type of calibrator best suitable for GMO quantification. The main question addressed in this review is whether reference materials and calibrators should be matrix based or whether pure DNA analytes should be used for relative quantification in GMO analysis. The state of the art, including the advantages and drawbacks, of using DNA plasmid (compared to genomic DNA reference materials) as calibrators, is widely described. In addition, the influence of the genetic structure of seeds on real-time PCR quantitative results obtained for seed lots is discussed. The specific composition of a seed kernel, the mode of inheritance, and the ploidy level ensure that there is discordance between a GMO % expressed as a haploid genome equivalent and a GMO % based on numbers of seeds. This means that a threshold fixed as a percentage of seeds cannot be used as such for RT-PCR. All critical points that affect the expression of the GMO content in seeds are discussed in this paper.

A Tissue-Specific Approach to the Analysis of Metabolic Changes in Caenorhabditis elegans

PubMed Central

Pujol, Claire; Ipsen, Sabine; Brodesser, Susanne; Mourier, Arnaud; Tolnay, Markus; Frank, Stephan; Trifunović, Aleksandra

2011-01-01

The majority of metabolic principles are evolutionarily conserved from nematodes to humans. Caenorhabditis elegans has widely accelerated the discovery of new genes important to maintain organismic metabolic homeostasis. Various methods exist to assess the metabolic state in worms, yet they often require large animal numbers and tend to be performed as bulk analyses of whole worm homogenates, thereby largely precluding a detailed studies of metabolic changes in specific worm tissues. Here, we have adapted well-established histochemical methods for the use on C. elegans fresh frozen sections and demonstrate their validity for analyses of morphological and metabolic changes on tissue level in wild type and various mutant strains. We show how the worm presents on hematoxylin and eosin (H&E) stained sections and demonstrate their usefulness in monitoring and the identification of morphological abnormalities. In addition, we demonstrate how Oil-Red-O staining on frozen worm cross-sections permits quantification of lipid storage, avoiding the artifact-prone fixation and permeabilization procedures of traditional whole-mount protocols. We also adjusted standard enzymatic stains for respiratory chain subunits (NADH, SDH, and COX) to monitor metabolic states of various C. elegans tissues. In summary, the protocols presented here provide technical guidance to obtain robust, reproducible and quantifiable tissue-specific data on worm morphology as well as carbohydrate, lipid and mitochondrial energy metabolism that cannot be obtained through traditional biochemical bulk analyses of worm homogenates. Furthermore, analysis of worm cross-sections overcomes the common problem with quantification in three-dimensional whole-mount specimens. PMID:22162770

quantGenius: implementation of a decision support system for qPCR-based gene quantification.

PubMed

Baebler, Špela; Svalina, Miha; Petek, Marko; Stare, Katja; Rotter, Ana; Pompe-Novak, Maruša; Gruden, Kristina

2017-05-25

Quantitative molecular biology remains a challenge for researchers due to inconsistent approaches for control of errors in the final results. Due to several factors that can influence the final result, quantitative analysis and interpretation of qPCR data are still not trivial. Together with the development of high-throughput qPCR platforms, there is a need for a tool allowing for robust, reliable and fast nucleic acid quantification. We have developed "quantGenius" ( http://quantgenius.nib.si ), an open-access web application for a reliable qPCR-based quantification of nucleic acids. The quantGenius workflow interactively guides the user through data import, quality control (QC) and calculation steps. The input is machine- and chemistry-independent. Quantification is performed using the standard curve approach, with normalization to one or several reference genes. The special feature of the application is the implementation of user-guided QC-based decision support system, based on qPCR standards, that takes into account pipetting errors, assay amplification efficiencies, limits of detection and quantification of the assays as well as the control of PCR inhibition in individual samples. The intermediate calculations and final results are exportable in a data matrix suitable for further statistical analysis or visualization. We additionally compare the most important features of quantGenius with similar advanced software tools and illustrate the importance of proper QC system in the analysis of qPCR data in two use cases. To our knowledge, quantGenius is the only qPCR data analysis tool that integrates QC-based decision support and will help scientists to obtain reliable results which are the basis for biologically meaningful data interpretation.

Selection and validation of reference genes for miRNA expression studies during porcine pregnancy.

PubMed

Wessels, Jocelyn M; Edwards, Andrew K; Zettler, Candace; Tayade, Chandrakant

2011-01-01

MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based

In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides

PubMed Central

2017-01-01

Collagen, the major structural component of nearly all mammalian tissues, undergoes extensive proteolytic remodeling during developmental states and a variety of life-threatening diseases such as cancer, myocardial infarction, and fibrosis. While degraded collagen could be an important marker of tissue damage, it is difficult to detect and target using conventional tools. Here, we show that a designed peptide (collagen hybridizing peptide: CHP), which specifically hybridizes to the degraded, unfolded collagen chains, can be used to image degraded collagen and inform tissue remodeling activity in various tissues: labeled with 5-carboxyfluorescein and biotin, CHPs enabled direct localization and quantification of collagen degradation in isolated tissues within pathologic states ranging from osteoarthritis and myocardial infarction to glomerulonephritis and pulmonary fibrosis, as well as in normal tissues during developmental programs associated with embryonic bone formation and skin aging. The results indicate the general correlation between the level of collagen remodeling and the amount of denatured collagen in tissue and show that the CHP probes can be used across species and collagen types, providing a versatile tool for not only pathology and developmental biology research but also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery in various animal models. PMID:28877431

Mesenchymal Stem Cell Levels of Human Spinal Tissues.

PubMed

Harris, Liam; Vangsness, C Thomas

2018-05-01

Systematic review. The aim of this study was to investigate, quantify, compare, and compile the various mesenchymal stem cell (MSC) tissue sources within human spinal tissues to act as a compendium for clinical and research application. Recent years have seen a dramatic increase in academic and clinical understanding of human MSCs. Previously limited to cells isolated from bone marrow, the past decade has illicited the characterization and isolation of human MSCs from adipose, bone marrow, synovium, muscle, periosteum, peripheral blood, umbilical cord, placenta, and numerous other tissues. As researchers explore practical applications of cells in these tissues, the absolute levels of MSCs in specific spinal tissue will be critical to guide future research. The PubMED, MEDLINE, EMBASE, and Cochrane databases were searched for articles relating to the harvest, characterization, isolation, and quantification of human MSCs from spinal tissues. Selected articles were examined for relevant data, categorized according to type of spinal tissue, and when possible, standardized to facilitate comparisons between sites. Human MSC levels varied widely between spinal tissues. Yields for intervertebral disc demonstrated roughly 5% of viable cells to be positive for MSC surface markers. Cartilage endplate cells yielded 18,500 to 61,875 cells/0.8 mm thick sample of cartilage end plate. Ligamentum flavum yielded 250,000 to 500,000 cells/g of tissue. Annulus fibrosus fluorescence activated cell sorting treatment found 29% of cells positive for MSC marker Stro-1. Nucleus pulposus yielded mean tissue samples of 40,584 to 234,137 MSCs per gram of tissue. Numerous tissues within and surrounding the spine represent a consistent and reliable source for the harvest and isolation of human MSCs. Among the tissues of the spine, the annulus fibrosus and ligamentum flavum each offer considerable levels of MSCs, and may prove comparable to that of bone marrow. 5.

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

PubMed

Rodriguez-Canales, Jaime; Hanson, Jeffrey C; Hipp, Jason D; Balis, Ulysses J; Tangrea, Michael A; Emmert-Buck, Michael R; Bova, G Steven

2013-01-01

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This updated chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high-quality, appropriately anatomically tagged scientific results. Improvement in this area will significantly increase life science quality and productivity. The chapter is divided into introduction, materials, protocols, and notes subheadings. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this chapter, readers are advised to read through the entire

Iterative fitting method for the evaluation and quantification of PAES spectra

NASA Astrophysics Data System (ADS)

Zimnik, Samantha; Hackenberg, Mathias; Hugenschmidt, Christoph

2017-01-01

The elemental composition of surfaces is of great importance for the understanding of many surface processes such as catalysis. For a reliable analysis and a comparison of results, the quantification of the measured data is indispensable. Positron annihilation induced Auger Electron Spectroscopy (PAES) is a spectroscopic technique that measures the elemental composition with outstanding surface sensitivity, but up to now, no standardized evaluation procedure for PAES spectra is available. In this paper we present a new approach for the evaluation of PAES spectra of compounds, using the spectra obtained for the pure elements as reference. The measured spectrum is then fitted by a linear combination of the reference spectra by varying their intensities. The comparison of the results of the fitting routine with a calculation of the full parameter range shows an excellent agreement. We present the results of the new analysis method to evaluate the PAES spectra of sub-monolayers of Ni on a Pd substrate.

Quantification of various growth factors in different demineralized bone matrix preparations.

PubMed

Wildemann, B; Kadow-Romacker, A; Haas, N P; Schmidmaier, G

2007-05-01

Besides autografts, allografts, and synthetic materials, demineralized bone matrix (DBM) is used for bone defect filling and treatment of non-unions. Different DBM formulations are introduced in clinic since years. However, little is known about the presents and quantities of growth factors in DBM. Aim of the present study was the quantification of eight growth factors important for bone healing in three different "off the shelf" DBM formulations, which are already in human use: DBX putty, Grafton DBM putty, and AlloMatrix putty. All three DBM formulations are produced from human donor tissue but they differ in the substitutes added. From each of the three products 10 different lots were analyzed. Protein was extracted from the samples with Guanidine HCL/EDTA method and human ELISA kits were used for growth factor quantification. Differences between the three different products were seen in total protein contend and the absolute growth factor values but also a large variability between the different lots was found. The order of the growth factors, however, is almost comparable between the materials. In the three investigated materials FGF basic and BMP-4 were not detectable in any analyzed sample. BMP-2 revealed the highest concentration extractable from the samples with approximately 3.6 microg/g tissue without a significant difference between the three DBM formulations. In DBX putty significantly more TGF-beta1 and FGFa were measurable compared to the two other DBMs. IGF-I revealed the significantly highest value in the AlloMatrix and PDGF in Grafton. No differences were accessed for VEGF. Due to the differences in the growth factor concentration between the individual samples, independently from the product formulation, further analyzes are required to optimize the clinical outcome of the used demineralized bone matrix. Copyright 2006 Wiley Periodicals, Inc.

43 CFR 11.71 - Quantification phase-service reduction quantification.

Code of Federal Regulations, 2011 CFR

2011-10-01

...-discharge-or-release condition. (c) Contents of the quantification. The following factors should be included...; and (6) Factors identified in the specific guidance in paragraphs (h), (i), (j), (k), and (l) of this section dealing with the different kinds of natural resources. (d) Selection of resources, services, and...

43 CFR 11.71 - Quantification phase-service reduction quantification.

Code of Federal Regulations, 2010 CFR

2010-10-01

...-discharge-or-release condition. (c) Contents of the quantification. The following factors should be included...; and (6) Factors identified in the specific guidance in paragraphs (h), (i), (j), (k), and (l) of this section dealing with the different kinds of natural resources. (d) Selection of resources, services, and...

An accurate proteomic quantification method: fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry.

PubMed

Liu, Junyan; Liu, Yang; Gao, Mingxia; Zhang, Xiangmin

2012-08-01

A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography-based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1-10(5) ) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot

NASA Astrophysics Data System (ADS)

Prentice, Boone M.; Chumbley, Chad W.; Caprioli, Richard M.

2017-01-01

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R2) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.

A novel method for quantification of beam's-eye-view tumor tracking performance.

PubMed

Hu, Yue-Houng; Myronakis, Marios; Rottmann, Joerg; Wang, Adam; Morf, Daniel; Shedlock, Daniel; Baturin, Paul; Star-Lack, Josh; Berbeco, Ross

2017-11-01

In-treatment imaging using an electronic portal imaging device (EPID) can be used to confirm patient and tumor positioning. Real-time tumor tracking performance using current digital megavolt (MV) imagers is hindered by poor image quality. Novel EPID designs may help to improve quantum noise response, while also preserving the high spatial resolution of the current clinical detector. Recently investigated EPID design improvements include but are not limited to multi-layer imager (MLI) architecture, thick crystalline and amorphous scintillators, and phosphor pixilation and focusing. The goal of the present study was to provide a method of quantitating improvement in tracking performance as well as to reveal the physical underpinnings of detector design that impact tracking quality. The study employs a generalizable ideal observer methodology for the quantification of tumor tracking performance. The analysis is applied to study both the effect of increasing scintillator thickness on a standard, single-layer imager (SLI) design as well as the effect of MLI architecture on tracking performance. The present study uses the ideal observer signal-to-noise ratio (d') as a surrogate for tracking performance. We employ functions which model clinically relevant tasks and generalized frequency-domain imaging metrics to connect image quality with tumor tracking. A detection task for relevant Cartesian shapes (i.e., spheres and cylinders) was used to quantitate trackability of cases employing fiducial markers. Automated lung tumor tracking algorithms often leverage the differences in benign and malignant lung tissue textures. These types of algorithms (e.g., soft-tissue localization - STiL) were simulated by designing a discrimination task, which quantifies the differentiation of tissue textures, measured experimentally and fit as a power-law in trend (with exponent β) using a cohort of MV images of patient lungs. The modeled MTF and NPS were used to investigate the effect of

Quantitative Analysis of Tissue Samples by Combining iTRAQ Isobaric Labeling with Selected/Multiple Reaction Monitoring (SRM/MRM).

PubMed

Narumi, Ryohei; Tomonaga, Takeshi

2016-01-01

Mass spectrometry-based phosphoproteomics is an indispensible technique used in the discovery and quantification of phosphorylation events on proteins in biological samples. The application of this technique to tissue samples is especially useful for the discovery of biomarkers as well as biological studies. We herein describe the application of a large-scale phosphoproteome analysis and SRM/MRM-based quantitation to develop a strategy for the systematic discovery and validation of biomarkers using tissue samples.

Correlation of X-ray computed tomography with quantitative nuclear magnetic resonance methods for pre-clinical measurement of adipose and lean tissues in living mice.

PubMed

Metzinger, Matthew N; Miramontes, Bernadette; Zhou, Peng; Liu, Yueying; Chapman, Sarah; Sun, Lucy; Sasser, Todd A; Duffield, Giles E; Stack, M Sharon; Leevy, W Matthew

2014-10-08

Numerous obesity studies have coupled murine models with non-invasive methods to quantify body composition in longitudinal experiments, including X-ray computed tomography (CT) or quantitative nuclear magnetic resonance (QMR). Both microCT and QMR have been separately validated with invasive techniques of adipose tissue quantification, like post-mortem fat extraction and measurement. Here we report a head-to-head study of both protocols using oil phantoms and mouse populations to determine the parameters that best align CT data with that from QMR. First, an in vitro analysis of oil/water mixtures was used to calibrate and assess the overall accuracy of microCT vs. QMR data. Next, experiments were conducted with two cohorts of living mice (either homogenous or heterogeneous by sex, age and genetic backgrounds) to assess the microCT imaging technique for adipose tissue segmentation and quantification relative to QMR. Adipose mass values were obtained from microCT data with three different resolutions, after which the data were analyzed with different filter and segmentation settings. Strong linearity was noted between the adipose mass values obtained with microCT and QMR, with optimal parameters and scan conditions reported herein. Lean tissue (muscle, internal organs) was also segmented and quantified using the microCT method relative to the analogous QMR values. Overall, the rigorous calibration and validation of the microCT method for murine body composition, relative to QMR, ensures its validity for segmentation, quantification and visualization of both adipose and lean tissues.

Framework for hyperspectral image processing and quantification for cancer detection during animal tumor surgery.

PubMed

Lu, Guolan; Wang, Dongsheng; Qin, Xulei; Halig, Luma; Muller, Susan; Zhang, Hongzheng; Chen, Amy; Pogue, Brian W; Chen, Zhuo Georgia; Fei, Baowei

2015-01-01

Hyperspectral imaging (HSI) is an imaging modality that holds strong potential for rapid cancer detection during image-guided surgery. But the data from HSI often needs to be processed appropriately in order to extract the maximum useful information that differentiates cancer from normal tissue. We proposed a framework for hyperspectral image processing and quantification, which includes a set of steps including image preprocessing, glare removal, feature extraction, and ultimately image classification. The framework has been tested on images from mice with head and neck cancer, using spectra from 450- to 900-nm wavelength. The image analysis computed Fourier coefficients, normalized reflectance, mean, and spectral derivatives for improved accuracy. The experimental results demonstrated the feasibility of the hyperspectral image processing and quantification framework for cancer detection during animal tumor surgery, in a challenging setting where sensitivity can be low due to a modest number of features present, but potential for fast image classification can be high. This HSI approach may have potential application in tumor margin assessment during image-guided surgery, where speed of assessment may be the dominant factor.

Framework for hyperspectral image processing and quantification for cancer detection during animal tumor surgery

NASA Astrophysics Data System (ADS)

Lu, Guolan; Wang, Dongsheng; Qin, Xulei; Halig, Luma; Muller, Susan; Zhang, Hongzheng; Chen, Amy; Pogue, Brian W.; Chen, Zhuo Georgia; Fei, Baowei

2015-12-01

Hyperspectral imaging (HSI) is an imaging modality that holds strong potential for rapid cancer detection during image-guided surgery. But the data from HSI often needs to be processed appropriately in order to extract the maximum useful information that differentiates cancer from normal tissue. We proposed a framework for hyperspectral image processing and quantification, which includes a set of steps including image preprocessing, glare removal, feature extraction, and ultimately image classification. The framework has been tested on images from mice with head and neck cancer, using spectra from 450- to 900-nm wavelength. The image analysis computed Fourier coefficients, normalized reflectance, mean, and spectral derivatives for improved accuracy. The experimental results demonstrated the feasibility of the hyperspectral image processing and quantification framework for cancer detection during animal tumor surgery, in a challenging setting where sensitivity can be low due to a modest number of features present, but potential for fast image classification can be high. This HSI approach may have potential application in tumor margin assessment during image-guided surgery, where speed of assessment may be the dominant factor.

Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses.

PubMed

Wang, Ming-Le; Li, Qing-Hui; Xin, Hua-Hong; Chen, Xuan; Zhu, Xu-Jun; Li, Xing-Hui

2017-01-01

Tea plants [Camellia sinensis (L.) O. Kuntze] are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants.

Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses

PubMed Central

Wang, Ming-Le; Li, Qing-Hui; Xin, Hua-Hong; Chen, Xuan; Zhu, Xu-Jun

2017-01-01

Tea plants [Camellia sinensis (L.) O. Kuntze] are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants. PMID:28453515

Effect of carbon monoxide on gene expression in cerebrocortical astrocytes: Validation of reference genes for quantitative real-time PCR.

PubMed

Oliveira, Sara R; Vieira, Helena L A; Duarte, Carlos B

2015-09-15

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as cytoprotection or inflammation. The accurate assessment of changes in gene expression depends on the selection of adequate internal reference gene(s). Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to this gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of CO on gene expression in cultured cerebrocortical astrocytes. The cells were exposed to CO by treatment with CORM-A1 (CO releasing molecule A1) and four different algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to evaluate the stability of eight putative reference genes. Our results indicate that Gapdh (glyceraldehyde-3-phosphate dehydrogenase) together with Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha (Succinate Dehydrogenase Complex, Subunit A), Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-A1 for up to 60 min. For validation, we analyzed the effect of CO on the expression of Bdnf and bcl-2. Different results were obtained, depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. These findings highlight the need for a proper and accurate selection of the reference genes used in the quantification of qRT-PCR results

Detection and quantification of pestivirus in experimentally infected pregnant ewes and their progeny.

PubMed

Hurtado, Ana; Sanchez, Isbene; Bastida, Felix; Minguijón, Esmeralda; Juste, Ramón A; García-Pérez, Ana L

2009-11-05

Border disease virus (BDV) causes important reproductive losses, and eradication strategies focus on the identification and removal of persistently infected animals arising after in uterine infection. BDV infection dynamics were studied in 13 ewes experimentally infected with BDV-4 genotype at 3 phases of pregnancy [days 108 (group A), 76 (group B) and 55 (group C)] by quantification of viral RNA in blood collected on days -1 to parturition using quantitative real-time RT-PCR (qRT-PCR). Viral RNA loads were also measured in blood/foetal fluid and tissue samples from their offspring at lambing (3 foetuses, 7 stillborns, 15 lambs). qRT-PCR results were compared with those obtained by conventional RT-PCR and used to predict persistent infections. Viral RNA was detected in the ewes between days 2-15 p.i. The viraemia reached its highest peak between days 6-7 p.i. with a second peak at days 11-12 p.i. qRT-PCR was significantly faster to perform (less than 1 h) than conventional RT-PCR and detected BDV RNA in more ewes, being detection more continuous and prolonged in time. The virus was detected in peripheral blood in a higher percentage of lambs than in tissues, where differences in viral genome copies were more marked. Skin and cerebral cortex showed the highest viral RNA loads, and spleen and spinal cord the lowest. High viral RNA loads were observed in several animals in group B and all in group C, infected during middle and early foetal development, respectively, but also in one lamb from group A, infected during late foetal development. Serology and viral genome copy number estimates in blood and tissues were used to establish a quantitative cut-off threshold for transient viraemia. Viral RNA quantification showed potential for the discrimination between persistent infections and transient viraemia using single-time point blood sampling and raised questions regarding foetal immune system development and the occurrence of persistent infections.

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)

USDA-ARS?s Scientific Manuscript database

Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a...

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

PubMed

Chida, Junji; Kido, Hiroshi

2014-01-01

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

Coordinating cell and tissue behavior during zebrafish neural tube morphogenesis.

PubMed

Araya, Claudio; Ward, Laura C; Girdler, Gemma C; Miranda, Miguel

2016-03-01

The development of a vertebrate neural epithelium with well-organized apico-basal polarity and a central lumen is essential for its proper function. However, how this polarity is established during embryonic development and the potential influence of surrounding signals and tissues on such organization has remained less understood. In recent years the combined superior transparency and genetics of the zebrafish embryo has allowed for in vivo visualization and quantification of the cellular and molecular dynamics that govern neural tube structure. Here, we discuss recent studies revealing how co-ordinated cell-cell interactions coupled with adjacent tissue dynamics are critical to regulate final neural tissue architecture. Furthermore, new findings show how the spatial regulation and timing of orientated cell division is key in defining precise lumen formation at the tissue midline. In addition, we compare zebrafish neurulation with that of amniotes and amphibians in an attempt to understand the conserved cellular mechanisms driving neurulation and resolve the apparent differences among animals. Zebrafish neurulation not only offers fundamental insights into early vertebrate brain development but also the opportunity to explore in vivo cell and tissue dynamics during complex three-dimensional animal morphogenesis. © 2015 Wiley Periodicals, Inc.

Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.

PubMed

Castro, Simone Vieira; Carvalho, Adeline Andrade; Silva, Cleidson Manoel Gomes; Santos, Francielli Weber; Campello, Cláudio Cabral; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

2014-10-01

The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

Quantitative micro-elastography: imaging of tissue elasticity using compression optical coherence elastography

PubMed Central

Kennedy, Kelsey M.; Chin, Lixin; McLaughlin, Robert A.; Latham, Bruce; Saunders, Christobel M.; Sampson, David D.; Kennedy, Brendan F.

2015-01-01

Probing the mechanical properties of tissue on the microscale could aid in the identification of diseased tissues that are inadequately detected using palpation or current clinical imaging modalities, with potential to guide medical procedures such as the excision of breast tumours. Compression optical coherence elastography (OCE) maps tissue strain with microscale spatial resolution and can delineate microstructural features within breast tissues. However, without a measure of the locally applied stress, strain provides only a qualitative indication of mechanical properties. To overcome this limitation, we present quantitative micro-elastography, which combines compression OCE with a compliant stress sensor to image tissue elasticity. The sensor consists of a layer of translucent silicone with well-characterized stress-strain behaviour. The measured strain in the sensor is used to estimate the two-dimensional stress distribution applied to the sample surface. Elasticity is determined by dividing the stress by the strain in the sample. We show that quantification of elasticity can improve the ability of compression OCE to distinguish between tissues, thereby extending the potential for inter-sample comparison and longitudinal studies of tissue elasticity. We validate the technique using tissue-mimicking phantoms and demonstrate the ability to map elasticity of freshly excised malignant and benign human breast tissues. PMID:26503225

Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip.

PubMed

Li, Wenbin; Abad, Jorge A; French-Monar, Ronald D; Rascoe, John; Wen, Aimin; Gudmestad, Neil C; Secor, Gary A; Lee, Ing-Ming; Duan, Yongping; Levy, Laurene

2009-07-01

The new Liberibacter species, 'Candidatus Liberibacter solanacearum' (Lso) recently associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand, was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the USA. A species-specific primer LsoF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lso in infected samples. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plants, which could be used to reliably access the DNA extraction quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither the qPCR nor the cPCR using the primer and/or probe sets with LsoF reacted with other Liberibacter species infecting citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target 16S rDNA templates per reaction for field samples. Lso was readily detected and quantified in various tissues of ZC-affected potato plants collected from fields in Texas. A thorough but uneven colonization of Lso was revealed in various tissues of potato plants. The highest Lso populations were about 3x10(8) genomes/g tissue in the root, which were 3-order higher than those in the above-ground tissues of potato plants. The Lso bacterial populations were normally distributed across the ZC-affected potato plants collected from fields in Texas, with 60% of ZC-affected potato plants harboring an average Lso population from 10(5) to 10(6) genomes/g tissue, 4% of plants hosting above 10(7) Lso genomes/g tissue, and 8% of plants holding below 10(3) Lso genomes/g tissue. The rapid, sensitive, specific and reliable multiplex qPCR showed its potential to become a powerful tool for early detection and quantification of the new Liberibacter species associated

Closed-reference metatranscriptomics enables in planta profiling of putative virulence activities in the grapevine trunk disease complex.

PubMed

Morales-Cruz, Abraham; Allenbeck, Gabrielle; Figueroa-Balderas, Rosa; Ashworth, Vanessa E; Lawrence, Daniel P; Travadon, Renaud; Smith, Rhonda J; Baumgartner, Kendra; Rolshausen, Philippe E; Cantu, Dario

2018-02-01

Grapevines, like other perennial crops, are affected by so-called 'trunk diseases', which damage the trunk and other woody tissues. Mature grapevines typically contract more than one trunk disease and often multiple grapevine trunk pathogens (GTPs) are recovered from infected tissues. The co-existence of different GTP species in complex and dynamic microbial communities complicates the study of the molecular mechanisms underlying disease development, especially under vineyard conditions. The objective of this study was to develop and optimize a community-level transcriptomics (i.e. metatranscriptomics) approach that could monitor simultaneously the virulence activities of multiple GTPs in planta. The availability of annotated genomes for the most relevant co-infecting GTPs in diseased grapevine wood provided the unprecedented opportunity to generate a multi-species reference for the mapping and quantification of DNA and RNA sequencing reads. We first evaluated popular sequence read mappers using permutations of multiple simulated datasets. Alignment parameters of the selected mapper were optimized to increase the specificity and sensitivity for its application to metagenomics and metatranscriptomics analyses. Initial testing on grapevine wood experimentally inoculated with individual GTPs confirmed the validity of the method. Using naturally infected field samples expressing a variety of trunk disease symptoms, we show that our approach provides quantitative assessments of species composition, as well as genome-wide transcriptional profiling of potential virulence factors, namely cell wall degradation, secondary metabolism and nutrient uptake for all co-infecting GTPs. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Quantification of polyhydroxyalkanoates in mixed and pure cultures biomass by Fourier transform infrared spectroscopy: comparison of different approaches.

PubMed

Isak, I; Patel, M; Riddell, M; West, M; Bowers, T; Wijeyekoon, S; Lloyd, J

2016-08-01

Fourier transform infrared (FTIR) spectroscopy was used in this study for the rapid quantification of polyhydroxyalkanoates (PHA) in mixed and pure culture bacterial biomass. Three different statistical analysis methods (regression, partial least squares (PLS) and nonlinear) were applied to the FTIR data and the results were plotted against the PHA values measured with the reference gas chromatography technique. All methods predicted PHA content in mixed culture biomass with comparable efficiency, indicated by similar residuals values. The PHA in these cultures ranged from low to medium concentration (0-44 wt% of dried biomass content). However, for the analysis of the combined mixed and pure culture biomass with PHA concentration ranging from low to high (0-93% of dried biomass content), the PLS method was most efficient. This paper reports, for the first time, the use of a single calibration model constructed with a combination of mixed and pure cultures covering a wide PHA range, for predicting PHA content in biomass. Currently no one universal method exists for processing FTIR data for polyhydroxyalkanoates (PHA) quantification. This study compares three different methods of analysing FTIR data for quantification of PHAs in biomass. A new data-processing approach was proposed and the results were compared against existing literature methods. Most publications report PHA quantification of medium range in pure culture. However, in our study we encompassed both mixed and pure culture biomass containing a broader range of PHA in the calibration curve. The resulting prediction model is useful for rapid quantification of a wider range of PHA content in biomass. © 2016 The Society for Applied Microbiology.

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

NASA Astrophysics Data System (ADS)

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

2016-04-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

Single 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

Combined fluorescence and reflectance spectroscopy for in vivo quantification of cancer biomarkers in low- and high-grade glioma surgery

NASA Astrophysics Data System (ADS)

Valdés, Pablo A.; Kim, Anthony; Leblond, Frederic; Conde, Olga M.; Harris, Brent T.; Paulsen, Keith D.; Wilson, Brian C.; Roberts, David W.

2011-11-01

Biomarkers are indicators of biological processes and hold promise for the diagnosis and treatment of disease. Gliomas represent a heterogeneous group of brain tumors with marked intra- and inter-tumor variability. The extent of surgical resection is a significant factor influencing post-surgical recurrence and prognosis. Here, we used fluorescence and reflectance spectral signatures for in vivo quantification of multiple biomarkers during glioma surgery, with fluorescence contrast provided by exogenously-induced protoporphyrin IX (PpIX) following administration of 5-aminolevulinic acid. We performed light-transport modeling to quantify multiple biomarkers indicative of tumor biological processes, including the local concentration of PpIX and associated photoproducts, total hemoglobin concentration, oxygen saturation, and optical scattering parameters. We developed a diagnostic algorithm for intra-operative tissue delineation that accounts for the combined tumor-specific predictive capabilities of these quantitative biomarkers. Tumor tissue delineation achieved accuracies of up to 94% (specificity = 94%, sensitivity = 94%) across a range of glioma histologies beyond current state-of-the-art optical approaches, including state-of-the-art fluorescence image guidance. This multiple biomarker strategy opens the door to optical methods for surgical guidance that use quantification of well-established neoplastic processes. Future work would seek to validate the predictive power of this proof-of-concept study in a separate larger cohort of patients.

Quantification of osteolytic bone lesions in a preclinical rat trial

NASA Astrophysics Data System (ADS)

Fränzle, Andrea; Bretschi, Maren; Bäuerle, Tobias; Giske, Kristina; Hillengass, Jens; Bendl, Rolf

2013-10-01

In breast cancer, most of the patients who died, have developed bone metastasis as disease progression. Bone metastases in case of breast cancer are mainly bone destructive (osteolytic). To understand pathogenesis and to analyse response to different treatments, animal models, in our case rats, are examined. For assessment of treatment response to bone remodelling therapies exact segmentations of osteolytic lesions are needed. Manual segmentations are not only time-consuming but lack in reproducibility. Computerized segmentation tools are essential. In this paper we present an approach for the computerized quantification of osteolytic lesion volumes using a comparison to a healthy reference model. The presented qualitative and quantitative evaluation of the reconstructed bone volumes show, that the automatically segmented lesion volumes complete missing bone in a reasonable way.

Depth-resolved monitoring of analytes diffusion in ocular tissues

NASA Astrophysics Data System (ADS)

Larin, Kirill V.; Ghosn, Mohamad G.; Tuchin, Valery V.

2007-02-01

Optical coherence tomography (OCT) is a noninvasive imaging technique with high in-depth resolution. We employed OCT technique for monitoring and quantification of analyte and drug diffusion in cornea and sclera of rabbit eyes in vitro. Different analytes and drugs such as metronidazole, dexamethasone, ciprofloxacin, mannitol, and glucose solution were studied and whose permeability coefficients were calculated. Drug diffusion monitoring was performed as a function of time and as a function of depth. Obtained results suggest that OCT technique might be used for analyte diffusion studies in connective and epithelial tissues.

Automated flow quantification in valvular heart disease based on backscattered Doppler power analysis: implementation on matrix-array ultrasound imaging systems.

PubMed

Buck, Thomas; Hwang, Shawn M; Plicht, Björn; Mucci, Ronald A; Hunold, Peter; Erbel, Raimund; Levine, Robert A

2008-06-01

Cardiac ultrasound imaging systems are limited in the noninvasive quantification of valvular regurgitation due to indirect measurements and inaccurate hemodynamic assumptions. We recently demonstrated that the principle of integration of backscattered acoustic Doppler power times velocity can be used for flow quantification in valvular regurgitation directly at the vena contracta of a regurgitant flow jet. We now aimed to accomplish implementation of automated Doppler power flow analysis software on a standard cardiac ultrasound system utilizing novel matrix-array transducer technology with detailed description of system requirements, components and software contributing to the system. This system based on a 3.5 MHz, matrix-array cardiac ultrasound scanner (Sonos 5500, Philips Medical Systems) was validated by means of comprehensive experimental signal generator trials, in vitro flow phantom trials and in vivo testing in 48 patients with mitral regurgitation of different severity and etiology using magnetic resonance imaging (MRI) for reference. All measurements displayed good correlation to the reference values, indicating successful implementation of automated Doppler power flow analysis on a matrix-array ultrasound imaging system. Systematic underestimation of effective regurgitant orifice areas >0.65 cm(2) and volumes >40 ml was found due to currently limited Doppler beam width that could be readily overcome by the use of new generation 2D matrix-array technology. Automated flow quantification in valvular heart disease based on backscattered Doppler power can be fully implemented on board a routinely used matrix-array ultrasound imaging systems. Such automated Doppler power flow analysis of valvular regurgitant flow directly, noninvasively, and user independent overcomes the practical limitations of current techniques.

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

PubMed

Nelson, Bryant C; Sharpless, Katherine E

2003-01-29

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

Image-Based Quantification of Plant Immunity and Disease.

PubMed

Laflamme, Bradley; Middleton, Maggie; Lo, Timothy; Desveaux, Darrell; Guttman, David S

2016-12-01

Measuring the extent and severity of disease is a critical component of plant pathology research and crop breeding. Unfortunately, existing visual scoring systems are qualitative, subjective, and the results are difficult to transfer between research groups, while existing quantitative methods can be quite laborious. Here, we present plant immunity and disease image-based quantification (PIDIQ), a quantitative, semi-automated system to rapidly and objectively measure disease symptoms in a biologically relevant context. PIDIQ applies an ImageJ-based macro to plant photos in order to distinguish healthy tissue from tissue that has yellowed due to disease. It can process a directory of images in an automated manner and report the relative ratios of healthy to diseased leaf area, thereby providing a quantitative measure of plant health that can be statistically compared with appropriate controls. We used the Arabidopsis thaliana-Pseudomonas syringae model system to show that PIDIQ is able to identify both enhanced plant health associated with effector-triggered immunity as well as elevated disease symptoms associated with effector-triggered susceptibility. Finally, we show that the quantitative results provided by PIDIQ correspond to those obtained via traditional in planta pathogen growth assays. PIDIQ provides a simple and effective means to nondestructively quantify disease from whole plants and we believe it will be equally effective for monitoring disease on excised leaves and stems.

Apoplastic Sugar Extraction and Quantification from Wheat Leaves Infected with Biotrophic Fungi.

PubMed

Roman-Reyna, Veronica; Rathjen, John P

2017-01-01

Biotrophic fungi such as rusts modify the nutrient status of their hosts by extracting sugars. Hemibiotrophic and biotrophic fungi obtain nutrients from the cytoplasm of host cells and/or the apoplastic spaces. Uptake of nutrients from the cytoplasm is via intracellular hyphae or more complex structures such as haustoria. Apoplastic nutrients are taken up by intercellular hyphae. Overall the infection creates a sink causing remobilization of nutrients from local and distal tissues. The main mobile sugar in plants is sucrose which is absorbed via plant or fungal transporters once unloaded into the cytoplasm or the apoplast. Infection by fungal pathogens alters the apoplastic sugar contents and stimulates the influx of nutrients towards the site of infection as the host tissue transitions to sink. Quantification of solutes in the apoplast can help to understand the allocation of nutrients during infection. However, separation of apoplastic fluids from whole tissue is not straightforward and leakage from damaged cells can alter the results of the extraction. Here, we describe how variation in cytoplasmic contamination and infiltrated leaf volumes must be controlled when extracting apoplastic fluids from healthy and rust-infected wheat leaves. We show the importance of correcting the data for these parameters to measure sugar concentrations accurately.

Variability of cytokine gene expression in intestinal tissue and the impact of normalization with the use of reference genes.

PubMed

McGowan, Ian; Janocko, Laura; Burneisen, Shaun; Bhat, Anand; Richardson-Harman, Nicola

2015-01-01

To determine the intra- and inter-subject variability of mucosal cytokine gene expression in rectal biopsies from healthy volunteers and to screen cytokine and chemokine mRNA as potential biomarkers of mucosal inflammation. Rectal biopsies were collected from 8 participants (3 biopsies per participant) and 1 additional participant (10 biopsies). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to quantify IL-1β, IL-6, IL-12p40, IL-8, IFN-γ, MIP-1α, MIP-1β, RANTES, and TNF-α gene expression in the rectal tissue. The intra-assay, inter-biopsy and inter-subject variance was measured in the eight participants. Bootstrap re-sampling of the biopsy measurements was performed to determine the accuracy of gene expression data obtained for 10 biopsies obtained from one participant. Cytokines were both non-normalized and normalized using four reference genes (GAPDH, β-actin, β2 microglobulin, and CD45). Cytokine measurement accuracy was increased with the number of biopsy samples, per person; four biopsies were typically needed to produce a mean result within a 95% confidence interval of the subject's cytokine level approximately 80% of the time. Intra-assay precision (% geometric standard deviation) ranged between 8.2 and 96.9 with high variance between patients and even between different biopsies from the same patient. Variability was not greatly reduced with the use of reference genes to normalize data. The number of biopsy samples required to provide an accurate result varied by target although 4 biopsy samples per subject and timepoint, provided for >77% accuracy across all targets tested. Biopsies within the same subjects and between subjects had similar levels of variance while variance within a biopsy (intra-assay) was generally lower. Normalization of inflammatory cytokines against reference genes failed to consistently reduce variance. The accuracy and reliability of mRNA expression of inflammatory cytokines will set a ceiling

Consistency of signal intensity and T2* in frozen ex vivo heart muscle, kidney, and liver tissue.

PubMed

Kaye, Elena A; Josan, Sonal; Lu, Aiming; Rosenberg, Jarrett; Daniel, Bruce L; Pauly, Kim Butts

2010-03-01

To investigate tissue dependence of the MRI-based thermometry in frozen tissue by quantification and comparison of signal intensity and T2* of ex vivo frozen tissue of three different types: heart muscle, kidney, and liver. Tissue samples were frozen and imaged on a 0.5 Tesla MRI scanner with ultrashort echo time (UTE) sequence. Signal intensity and T2* were determined as the temperature of the tissue samples was decreased from room temperature to approximately -40 degrees C. Statistical analysis was performed for (-20 degrees C, -5 degrees C) temperature interval. The findings of this study demonstrate that signal intensity and T2* are consistent across three types of tissue for (-20 degrees C, -5 degrees C) temperature interval. Both parameters can be used to calculate a single temperature calibration curve for all three types of tissue and potentially in the future serve as a foundation for tissue-independent MRI-based thermometry.