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PubMed2016

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.

5-Methylcytosine↗
PubMed2025

Absolute quantification of the living skin microbiome overcomes relic-DNA bias and reveals specific patterns across volunteers.

BACKGROUND: As the first line of defense against external pathogens, the skin and its resident microbiota are responsible for protection and eubiosis. Innovations in DNA sequencing have significantly increased our knowledge of the skin microbiome. However, current characterizations do not discriminate between DNA from live cells and remnant DNA from dead organisms (relic DNA), resulting in a combined readout of all microorganisms that were and are currently present on the skin rather than the actual living population of the microbiome. Additionally, most methods lack the capability for absolute quantification of the microbial load on the skin, complicating the extrapolation of clinically relevant information. RESULTS: Here, we integrated relic-DNA depletion with shotgun metagenomics and bacterial load determination to quantify live bacterial cell abundances across different skin sites. Though we discovered up to 90% of microbial DNA from the skin to be relic DNA, we saw no significant effect of this on the relative abundances of taxa determined by shotgun sequencing. Relic-DNA depletion prior to sequencing strengthened underlying patterns between microbiomes across volunteers and reduced intraindividual similarity. We determined the absolute abundance and the fraction of population alive for several common skin taxa across body sites and found taxa-specific differential abundance of live bacteria across regions to be different from estimates generated by total DNA (live + dead) sequencing. CONCLUSIONS: Our results reveal the significant bias relic DNA has on the quantification of low biomass samples like the skin. The reduced intraindividual similarity across samples following relic-DNA depletion highlights the bias introduced by traditional (total DNA) sequencing in diversity comparisons across samples. The divergent levels of cell viability measured across different skin sites, along with the inconsistencies in taxa differential abundance determined by total vs live cell DNA sequencing, suggest an important hypothesis for certain sites being susceptible to pathogen infection. Overall, our study demonstrates a characterization of the skin microbiome that overcomes relic-DNA bias to provide a baseline for live microbiota that will further improve mechanistic studies of infection, disease progression, and the design of therapies for the skin. Video Abstract.

Humans↗
PubMed2002

Rapid quantification of murine ABC mRNAs by real time reverse transcriptase-polymerase chain reaction.

Several ATP-binding cassette (ABC) transporters are critically involved in cholesterol and phospholipid efflux, reverse cholesterol transport, and play an important role in the development of atherosclerosis. Quantification of ABC mRNA is important in studying the regulation of cellular cholesterol homeostasis and mechanisms related to the pathogenesis of atherosclerosis. We have developed a one-step real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for measuring mRNA levels of ABCA1, ABCG1, and ABCA2 in murine tissues using the TaqMan(TM) technology. It has significant methodological benefits when compared to classic Northern blotting or semi-quantitative RT-PCR analysis. Using this method, we found high expression levels of ABCA1 in liver and macrophages, and of ABCG1 in the brain and macrophages. The expression of ABCA1 and ABCG1 were further induced in macrophages loaded with acLDL. In contrast, ABCA2 was expressed exclusively in the brain with low expression levels in the macrophages. This method provides a rapid, highly sensitive, specific, and reproducible quantification of ABC mRNA, and can be performed with nanograms of total RNA sample, thus making it a superior method for studying the regulation of ABC transporters in cholesterol efflux and its role in the pathogenesis of atherosclerosis in murine models.

ATP-Binding Cassette Transporters↗
PubMed2003

Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride.

We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [3H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an approximately 40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation. The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.

Animals↗
PubMed2006

Highly sensitive quantification of 7alpha-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS.

We describe a highly sensitive and specific method for the quantification of serum 7alpha-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 mul) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha-picolinate), and then purified using a disposable C(18) cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 +/- 2.8 ng/ml, which coincided completely with the observed X(0) +/- SD = 23.3 +/- 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.

Calibration↗
PubMed2003

Prognostic value of minimal residual disease quantification by real-time reverse transcriptase polymerase chain reaction in patients with core binding factor leukemias.

PURPOSE: In patients with acute myeloblastic leukemia with t(8;21) or inv(16) aberrations (core binding factor [CBF] leukemias), minimal residual disease (MRD) can be sensitively detected during and after chemotherapy by use of molecular methods. However, the prognostic impact of qualitative MRD detection is still under debate. In this study, the prognostic value of MRD quantification in patients with CBF leukemias was assessed. PATIENTS AND METHODS: We quantified MRD at various time points during and after therapy by real-time reverse transcriptase polymerase chain reaction (RT-PCR) for AML1/MTG8 and CBFB/MYH11 in 37 patients with CBF leukemias treated within a multicenter trial. RESULTS: At initial diagnosis, the patients showed a heterogenous fusion gene expression relative to glyceraldehyde 3-phosphate dehydrogenase with a variation of more than two log steps. According to MRD status during/after therapy, two groups of patients were separated. Of the 26 patients who had MRD levels of less than 1% in relation to initial diagnosis at all time points tested after induction chemotherapy, only two experienced relapse after a median follow-up of 19 months. Of the 11 patients who had a sample with an MRD level >/= 1% at least at one time point after induction therapy, 10 experienced relapse, with a median remission duration of 10 months (P <.001). The median interval between the informative MRD sample and clinical relapse in these patients was 3 months. CONCLUSION: MRD quantification by real-time RT-PCR allows the identification of patients with a high risk of relapse among the CBF leukemias.

Adolescent↗
PubMed1999

HbF synthesis during stress erythropoiesis as determined by gamma-mRNA/non-alpha-mRNA quantification.

To determine whether a quantitative relationship exists between globin mRNAs and their translation product during stress erythropoiesis in infants with increased production of fetal hemoglobin (HbF), we measured and compared the relative amounts of the mRNAs of alpha-, beta-, and gamma-globins and their protein synthesis. The synthesis of globin in immature red cells was determined by the incorporation of [3H]leucine, followed by separation and quantification of the polypeptides by C4-reverse phase HPLC. The relative proportions of the mRNAs of the globins were determined by RNase protection assay. A comparison of blood samples from 17 infants expected to have increased production of HbF in relation to their developmental age (five infants of diabetic mothers, two infants with intrauterine growth retardation, one infant with bronchopulmonary dysplasia, and seven infants with cyanotic heart disease) revealed a very significant correlation (r2 = 0.994; p < 0.001) between the ratio of globin mRNAs encoding HbF ([gamma/(gamma + beta)] mRNAs) and the ratios of the de novo synthesis of HbF [gamma/(gamma + beta)]. When only the 10 infants who had increased HbF synthesis are included, the correlation remains unchanged (r2 = 0.997, p < 0.001). The data demonstrated that under conditions of erythropoietic stress, when HbF production is increased, there is a close relationship between the quantification of gamma-globin mRNA and gamma-globin synthesis. The usual methods of determining HbF synthesis can be replaced by globin mRNA determination, which can be performed rapidly with a minimal amount of blood.

Aging↗
PubMed2006

Acoustic-resonance spectrometry as a process analytical technology for the quantification of active pharmaceutical ingredient in semi-solids.

The purpose of this study was to demonstrate acoustic resonance spectrometry (ARS) as an alternative process analytical technology to near infrared (NIR) spectroscopy for the quantification of active pharmaceutical ingredient (API) in semi-solids such as creams, gels, ointments, and lotions. The ARS used for this research was an inexpensive instrument constructed from readily available parts. Acoustic-resonance spectra were collected with a frequency spectrum from 0 to 22.05 KHz. NIR data were collected from 1100 to 2500 nm. Using 1-point net analyte signal (NAS) calibration, NIR for the API (colloidal oatmeal [CO]) gave an r2 prediction accuracy of 0.971, and a standard error of performance (SEP) of 0.517%CO. ARS for the API resulted in an r2 of 0.983 and SEP of 0.317%CO. NAS calibration is compared with principal component regression. This research demonstrates that ARS can sometimes outperform NIR spectrometry and can be an effective analytical method for the quantification of API in semi-solids. ARS requires no sample preparation, provides larger penetration depths into lotions than optical techniques, and measures API concentrations faster and more accurately. These results suggest that ARS is a useful process analytical technology (PAT).

Acoustics↗
PubMed2002

Monoclonal anti-epidermal growth factor receptor (ior EGF/r3) antibody pharmacokinetic studies on nude mice I: a radio-receptor analysis applied to drug serum quantification.

Due to its antagonistic properties upon ligand-epidermal growth factor receptor (EGFr) interaction, the monoclonal antibody anti-EGFr ior EGF/r3 is considered a potential therapeutic agent against several epithelium-derived tumours. This paper affords further analysis of the relevant corporal interaction of this monoclonal antibody in terms of its pharmacodynamic properties, using nude mice, following a single bolus intravenous dose administration. The radio-receptor assay allows quantification of the serum ior EGF/r3 level. The dose selection procedure, according to the Kolmogorov-Smirnov test, suggested using doses of 12.5-16 mg kg(-1) for pharmacokinetic assessments. The experimental data were best fitted to a bi-exponential function (r2 = 0.985), through the classical two-compartment open modelling approach. The model selection was corroborated by the AKAIKE information criteria, and also the SCHWARTZ and ESTRIP test were used. The estimated pharmacokinetic parameters (e.g. t 1/2 beta = 34.65 h, Vc = 2.84 mL, Vss = 4.21 mL and CL = 0.09 mL h(-1)) bear out the strategies for the evaluation of the therapeutic application of this drug. Finally, the radio-receptor analysis has provided a rationale for the proposed serum monoclonal antibody ior EGF/r3 quantification to characterize its concentration-time course.

Animals↗
PubMed2001

Development and validation of a sensitive ELISA for quantification of secretory IgA in rat saliva and faeces.

Non-invasive measures of immunological markers are an attractive means of stress assessment in laboratory animals. Salivary IgA has been used successfully as a stress marker in the human, and several reports indicate the potential of secretory IgA as a non-invasive measure of stress in animals. The present paper describes the development of an ELISA using commercially available components for the quantification of rat IgA and validation of this assay for the quantification of rat secretory IgA in saliva and faeces. The concentration of IgA in rat saliva varied significantly between duplicate samples obtained from individual rats, and the viscosity and small total volume of rat saliva gave unsatisfactory results for IgA. Faecal IgA was present in high concentrations, and duplicate samples varied by only 2-3%. However, faecal IgA seemed less stable than IgA in other biological compartments, and this finding must be taken into consideration when using quantitative measurements of IgA as a marker of mucous humoral immune status.

Animals↗
PubMed1994

Quantification of atherosclerotic plaques in carotid arteries by three-dimensional ultrasound.

The aim of this study was to develop three-dimensional (3D) ultrasound imaging of the carotid arteries in order to reduce intraexaminer and interexaminer variabilities of volume measurements in follow-up scans of atherosclerotic plaques. The non-invasive quantification of human atherosclerosis by 3D ultrasound was performed using a three-dimensional computer based image reconstruction. The linear movements of the transducer were driven by a motor triggered by electrocardiography. The method included an option to change angle of insonation and a correction of skin contact adaptation. Repeated measurements were performed at weekly intervals in 70 patients. Variabilities and ranges of three newly developed evaluation procedures of plaque volumes (manual tracing, threshold procedure, watershed algorithm) were used. Intraexaminer variability varied between 2.8% (watershed algorithm) and 4.1% (threshold procedure). Interexaminer variability ranged from 4.2% (manual tracing) to 7.6% (threshold procedure), follow-up measurements from 5.2% (watershed algorithm) to 9.4% (threshold procedure). Pearson correlation coefficients of interexaminer agreement and also those between all three measurement procedures were highly significant (p < 0.01). We conclude that the newly developed 3D computer based image reconstruction allows repeated quantification of moderate atherosclerosis with a high reliability.

Arteriosclerosis↗
PubMed2005

An isotope effect on the comparative quantification of flavonoids by means of methylation-based stable isotope dilution coupled with capillary liquid chromatography/mass spectrometry.

Ionization suppression is a serious problem in liquid chromatography/mass spectrometry-based metabolomics, and stable isotope dilution-based comparative quantification is one of the most important methods of overcoming this problem. Herein, the use of [(13)C]-methylation-based stable isotope dilution for comparative quantification of flavonoids is demonstrated. This is in contrast to the equivalent deuterium labeling methylation method, which has an adverse isotope effect on reverse phase chromatography.

Artifacts↗
PubMed2005

Stable isotope dilution-based accurate comparative quantification of nitrogen-containing metabolites in Arabidopsis thaliana T87 cells using in vivo (15)N-isotope enrichment.

Stable isotope dilution-based comparative quantification of nitrogen-containing metabolites for highly sensitive and selective metabolomics was developed using liquid chromatography/mass spectrometry (LC/MS) and (15)N-isotope enrichment. We produced metabolically stable isotope-labeled Arabidopsis T87 cells by culturing with (15)N-labeled medium. We found that the growth of cells maintained in (15)N-labeled medium is very similar to the growth in normal medium, as evidenced by cell morphology, doubling time, and measurement of chlorophyll and carotenoid contents. Complete incorporation of (15)N in folate, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) in T87 cells was accomplished after culturing for 21 d. Accurate comparative quantification of folate, SAM, and SAH was established by means of LC/MS using the isotopomers of the target metabolites as internal standards. The within- and between-run assay coefficients of variation for the folate, SAM, and SAH levels were all less than 8.5%. Stable isotope labeling by nitrogen source in Arabidopsis T87 cell culture provided simple, inexpensive, and accurate amino acid profiling. This interesting new protocol is valuable for the study of dynamic changes in N-compound pools in cultured cells.

Arabidopsis↗
PubMed1991

[Estimation of portal pressure using the theory of quantification].

An attempt was made to estimate noninvasively portal pressure (PP) in patients with chronic liver disease, using the theory of quantification, a kind of multivariate analysis. Forty-one patients with liver cirrhosis and 22 patients with chronic hepatitis in whom hepatic venous catheterization had been performed were studied. Seventeen parameters (age, sex, mean blood pressure, red blood cell count, platelet count, prothrombin time, lactate dehydrogenase, alkaline phosphatase, total bilirubin, albumin, gamma-globulin, indocyanine green retention at 15 min, blood urea nitrogen, hepatomegaly, splenomegaly, ascites and edema) were selected for the estimation of PP. The estimated PP correlated significantly with the data obtained by hepatic venous catheterization with a high correlation coefficient of 0.835 (p less than 0.01). An investigation using the theory of quantification was also undertaken to determine which of the 17 parameters selected above was most useful in estimating PP. Among the 17 parameters indocyanine green retention at 15 min, red blood cell count, prothrombin time, hepatomegaly and splenomegaly seemed to contribute significantly to the estimation of PP. When the formula was applied to 31 successive patients with chronic liver disease (external samples), the correlation between the estimated and measured PP was 0.455 (p less than 0.01). These results indicate that the formula is clinically useful in estimating PP in patients with chronic liver disease.

Adult↗
PubMed2001

Quantification and facilitated comparison of von Willebrand factor multimer patterns by densitometry.

The analysis of von Willebrand factor (vWF) multimers is an important laboratory tool for distinguishing among the numerous subtypes of von Willebrand disease (vWD). Comparability and reproducibility of this method are insufficient; standardization and external references are pending. Interlaboratory comparison of results therefore may be difficult. We applied densitometry to obtain a reproducible quantification of vWF multimer patterns in healthy donors, patients with vWD variants, and factor VIII/vWF concentrates to improve the reproducibility and comparability of vWF multimer analysis. Multimers were separated and visualized luminographically on x-ray films. Films were scanned and evaluated by densitometry. The variation inherent in vWF multimer analysis and the range of the normal could be quantified. In vWD variants and factor VIII/vWF concentrates, densitometry could quantify and visualize alterations of vWF multimer patterns and facilitate their comparison. Densitometry permits a precise quantitative comparison of sample patterns to a reference plasma. It could be a valuable tool offering reproducible quantification and additional visualization of normal and pathologic vWF multimer patterns, facilitating their comparison and contributing to a standardization of vWF multimer analysis.

Adult↗
PubMed1997

Quantification of biochemical markers of bone turnover by kinetic measures of bone formation and resorption in young healthy females.

The quantification of biochemical markers of bone formation and resorption with kinetic measures of bone turnover is an essential step in their validation. Some biochemical markers have been validated by quantification against formation and resorption rates measured by calcium kinetics in adults with bone disease. However, none has been validated in healthy individuals who are undergoing skeletal growth and bone consolidation. Therefore, we have measured biochemical markers of bone formation (serum osteocalcin [OC], bone-specific alkaline phosphatase [BAP], and total alkaline phosphatase [ALP]) and resorption (serum tartrate resistant acid phosphatase [TRAP], urinary cross-linked N teleopeptides of type I collagen/creatinine [NTx/Cr], and hydroxyproline/creatinine [OHP/Cr]) in healthy females aged 11-32 years (n = 31) after an overnight fast to determine their relationship with bone formation (Vo+) and bone resorption (Vo-) as measured by calcium kinetics and balance. All biochemical markers were highly intercorrelated (r > 0.6, p < 0.001) as were Vo+ and Vo- (r = 0.91, p < 0.001). Highly significant correlations were present between bone formation measured by calcium kinetics (Vo+) and serum levels of bone biochemical markers (OC, r = 0.82, p = 0.001; ALP, r = 0.92, p = 0.001; and BAP, r = 0.90, p = 0.001) and between bone resorption measured by calcium kinetics (Vo-) and fasting serum levels and urine creatinine ratios of biochemical markers (TRAP, r = 0.77, p < 0.001; OHP/Cr, r = 0.79, p < 0.001; and NTx/Cr, r = 0.70, p < 0.001). Thus, biochemical markers of bone formation and resorption can be used to predict calcium kinetic rates during skeletal growth and the early years of formation of peak bone mass, ages at which strategies to build peak bone mass are important. Biochemical markers of formation and resorption are equally useful in predicting either the bone formation rate or the resorption rate.

Acid Phosphatase↗
PubMed2006

Apparent and inherent optical properties of turbid estuarine waters: measurements, empirical quantification relationships, and modeling.

Spectral measurements of remote-sensing reflectance (Rrs) and absorption coefficients carried out in three European estuaries (Gironde and Loire in France, Tamar in the UK) are presented and analyzed. Typical Rrs and absorption spectra are compared with typical values measured in coastal waters. The respective contributions of the water constituents, i.e., suspended sediments, colored dissolved organic matter, and phytoplankton (characterized by chlorophyll-a), are determined. The Rrs spectra are then reproduced with an optical model from the measured absorption coefficients and fitted backscattering coefficients. From Rrs ratios, empirical quantification relationships are established, reproduced, and explained from theoretical calculations. These quantification relationships were established from numerous field measurements and a reflectance model integrating the mean values of the water constituents' inherent optical properties. The model's sensitivity to the biogeochemical constituents and to their nature and composition is assessed.

Journal Article↗
PubMed2005

Improved understanding of factors contributing to quantification of anhydrate/hydrate powder mixtures.

Different spectroscopic approaches have proved to be excellent analytical tools for monitoring process-induced transformations of active pharmaceutical ingredients during pharmaceutical unit operations. In order to use these tools effectively, it is necessary to build calibration models that describe the relationship between the amount of each solid-state form of interest and the spectroscopic signal. In this study, near-infrared (NIR) and Raman spectroscopic methods have been evaluated for the quantification of hydrate and anhydrate forms in pharmaceutical powders. Process type spectrometers were used to collect the data and the role of the sampling procedure was examined. Multivariate regression models were compared with traditional univariate calibrations and special emphasis was placed on data treatment prior to multivariate modeling by partial least squares (PLS). It was found that the measured sample volume greatly affected the performance of the model whereby the calibrations were significantly improved by utilizing a larger sampling area. In addition, multivariate regression did not always improve the predictability of the data compared to univariate analysis. The data treatment prior to multivariate modeling had a significant influence on the quality of predictions with standard normal variate transformation generally proving to be the best preprocessing method. When the appropriate sampling techniques and data analysis methods were utilized, both NIR and Raman spectroscopy were found to be suitable methods for the quantification of anhydrate/hydrate in powder systems, and thus the method of choice will depend on the conditions in the process under investigation.

Computer Systems↗