Comparison between a high-resolution single-stage Orbitrap and a triple quadrupole mass spectrometer for quantitative analyses of drugs.

The capabilities of a high-resolution (HR), accurate mass spectrometer (Exactive-MS) operating in full scan MS mode was investigated for the quantitative LC/MS analysis of drugs in patients' plasma samples. A mass resolution of 50,000 (FWHM) at m/z 200 and a mass extracted window of 5 ppm around the theoretical m/z of each analyte were used to construct chromatograms for quantitation. The quantitative performance of the Exactive-MS was compared with that of a triple quadrupole mass spectrometer (TQ-MS), TSQ Quantum Discovery or Quantum Ultra, operating in the conventional selected reaction monitoring (SRM) mode. The study consisted of 17 therapeutic drugs including 8 antifungal agents (anidulafungin, caspofungin, fluconazole, itraconazole, hydroxyitraconazole posaconazole, voriconazole and voriconazole-N-oxide), 4 immunosuppressants (ciclosporine, everolimus, sirolimus and tacrolimus) and 5 protein kinase inhibitors (dasatinib, imatinib, nilotinib, sorafenib and sunitinib). The quantitative results obtained with HR-MS acquisition show comparable detection specificity, assay precision, accuracy, linearity and sensitivity to SRM acquisition. Importantly, HR-MS offers several benefits over TQ-MS technology: absence of SRM optimization, time saving when changing the analysis from one MS to another, more complete information of what is in the samples and easier troubleshooting. Our work demonstrates that U/HPLC coupled to Exactive HR-MS delivers comparable results to TQ-MS in routine quantitative drug analyses. Considering the advantages of HR-MS, these results suggest that, in the near future, there should be a shift in how routine quantitative analyses of small molecules, particularly for therapeutic drugs, are performed.

Heritability and quantitative genetic divergence of serotiny, a fire-persistence plant trait.

BACKGROUND AND AIMS: Although it is well known that fire acts as a selective pressure shaping plant phenotypes, there are no quantitative estimates of the heritability of any trait related to plant persistence under recurrent fires, such as serotiny. In this study, the heritability of serotiny in Pinus halepensis is calculated, and an evaluation is made as to whether fire has left a selection signature on the level of serotiny among populations by comparing the genetic divergence of serotiny with the expected divergence of neutral molecular markers (QST-FST comparison). METHODS: A common garden of P. halepensis was used, located in inland Spain and composed of 145 open-pollinated families from 29 provenances covering the entire natural range of P. halepensis in the Iberian Peninsula and Balearic Islands. Narrow-sense heritability (h(2)) and quantitative genetic differentiation among populations for serotiny (QST) were estimated by means of an 'animal model' fitted by Bayesian inference. In order to determine whether genetic differentiation for serotiny is the result of differential natural selection, QST estimates for serotiny were compared with FST estimates obtained from allozyme data. Finally, a test was made of whether levels of serotiny in the different provenances were related to different fire regimes, using summer rainfall as a proxy for fire regime in each provenance. KEY RESULTS: Serotiny showed a significant narrow-sense heritability (h(2)) of 0·20 (credible interval 0·09-0·40). Quantitative genetic differentiation among provenances for serotiny (QST = 0·44) was significantly higher than expected under a neutral process (FST = 0·12), suggesting adaptive differentiation. A significant negative relationship was found between the serotiny level of trees in the common garden and summer rainfall of their provenance sites. CONCLUSIONS: Serotiny is a heritable trait in P. halepensis, and selection acts on it, giving rise to contrasting serotiny levels among populations depending on the fire regime, and supporting the role of fire in generating genetic divergence for adaptive traits.

Benefits from ecological study methods to taxonomy of enchytraeids in southern Mata Atlântica

The objective of this work was to determine how taxonomy benefited from the ecological quantitative and site-based sampling methods in enchytraeids studies. Enchytraeids (small relatives of earthworms) were sampled in different phases of rain forest regeneration in the southern Mata Atlântica in Paraná, Brazil. The research combined ecological and taxonomic work, because enchytraeids are poorly studied and difficult to identify, and many new species were expected. The provision of large numbers of specimens enabled the test of species diagnoses by investigating the ranges of character variations in a larger series of specimens. Simplified species diagnoses adapted to the local conditions that allowed the identification of all specimens, juveniles included, were developed. Key characters and character states are presented for the three genera: Achaeta, Hemienchytraeus and Guaranidrilus. Among several new species, a rare species, possibly a remnant of the autochthonous forest fauna, was found and described.

Statistical significance of quantitative PCR.

BACKGROUND: PCR has the potential to detect and precisely quantify specific DNA sequences, but it is not yet often used as a fully quantitative method. A number of data collection and processing strategies have been described for the implementation of quantitative PCR. However, they can be experimentally cumbersome, their relative performances have not been evaluated systematically, and they often remain poorly validated statistically and/or experimentally. In this study, we evaluated the performance of known methods, and compared them with newly developed data processing strategies in terms of resolution, precision and robustness. RESULTS: Our results indicate that simple methods that do not rely on the estimation of the efficiency of the PCR amplification may provide reproducible and sensitive data, but that they do not quantify DNA with precision. Other evaluated methods based on sigmoidal or exponential curve fitting were generally of both poor resolution and precision. A statistical analysis of the parameters that influence efficiency indicated that it depends mostly on the selected amplicon and to a lesser extent on the particular biological sample analyzed. Thus, we devised various strategies based on individual or averaged efficiency values, which were used to assess the regulated expression of several genes in response to a growth factor. CONCLUSION: Overall, qPCR data analysis methods differ significantly in their performance, and this analysis identifies methods that provide DNA quantification estimates of high precision, robustness and reliability. These methods allow reliable estimations of relative expression ratio of two-fold or higher, and our analysis provides an estimation of the number of biological samples that have to be analyzed to achieve a given precision.

Improved detection of Rhodococcus coprophilus with a new quantitative PCR assay.

Agricultural practices, such as spreading liquid manure or the utilisation of land as animal pastures, can result in faecal contamination of water resources. Rhodococcus coprophilus is used in microbial source tracking to indicate animal faecal contamination in water. Methods previously described for detecting of R. coprophilus in water were neither sensitive nor specific. Therefore, the aim of this study was to design and validate a new quantitative polymerase chain reaction (qPCR) to improve the detection of R. coprophilus in water. The new PCR assay was based on the R. coprophilus 16S rRNA gene. The validation showed that the new approach was specific and sensitive for deoxyribunucleic acid from target host species. Compared with other PCR assays tested in this study, the detection limit of the new qPCR was between 1 and 3 log lower. The method, including a filtration step, was further validated and successfully used in a field investigation in Switzerland. Our work demonstrated that the new detection method is sensitive and robust to detect R. coprophilus in surface and spring water. Compared with PCR assays that are available in the literature or to the culture-dependent method, the new molecular approach improves the detection of R. coprophilus.

Iowa Fen Rapid Assessment Method for Wetlands v. 1 Quantitative Rating, 2016

Rapid assessment methods are valuable tools for collecting information about the quality and status of natural systems. However, they are not a substitute for detailed surveys of those systems. Users of this method should consider that the method may under-score or over-score the site that’s assessed, especially when the site is not a typical fen (i.e. a sedge meadow could score lower than fens, but in-fact be a relatively high quality sedge meadow). This assessment can be used throughout most of the spring, summer and fall period, however the ideal “index period” would be from late May through early October when native plant communities, as well as invasive species, are most apparent.

Comparison and validation of tissue modelization and statistical classification methods in T1-weighted MR brain images.

This paper presents a validation study on statistical nonsupervised brain tissue classification techniques in magnetic resonance (MR) images. Several image models assuming different hypotheses regarding the intensity distribution model, the spatial model and the number of classes are assessed. The methods are tested on simulated data for which the classification ground truth is known. Different noise and intensity nonuniformities are added to simulate real imaging conditions. No enhancement of the image quality is considered either before or during the classification process. This way, the accuracy of the methods and their robustness against image artifacts are tested. Classification is also performed on real data where a quantitative validation compares the methods' results with an estimated ground truth from manual segmentations by experts. Validity of the various classification methods in the labeling of the image as well as in the tissue volume is estimated with different local and global measures. Results demonstrate that methods relying on both intensity and spatial information are more robust to noise and field inhomogeneities. We also demonstrate that partial volume is not perfectly modeled, even though methods that account for mixture classes outperform methods that only consider pure Gaussian classes. Finally, we show that simulated data results can also be extended to real data.

Defining risk thresholds for a 10-year probability of hip fracture model that combines clinical risk factors and quantitative ultrasound: results using the EPISEM cohort.

Using a large prospective cohort of over 12,000 women, we determined 2 thresholds (high risk and low risk of hip fracture) to use in a 10-yr hip fracture probability model that we had previously described, a model combining the heel stiffness index measured by quantitative ultrasound (QUS) and a set of easily determined clinical risk factors (CRFs). The model identified a higher percentage of women with fractures as high risk than a previously reported risk score that combined QUS and CRF. In addition, it categorized women in a way that was quite consistent with the categorization that occurred using dual X-ray absorptiometry (DXA) and the World Health Organization (WHO) classification system; the 2 methods identified similar percentages of women with and without fractures in each of their 3 categories, but the 2 identified only in part the same women. Nevertheless, combining our composite probability model with DXA in a case findings strategy will likely further improve the detection of women at high risk of fragility hip fracture. We conclude that the currently proposed model may be of some use as an alternative to the WHO classification criteria for osteoporosis, at least when access to DXA is limited.

Quantitative modelling of transcription factor binding sites

Abstract One of the most important issues in molecular biology is to understand regulatory mechanisms that control gene expression. Gene expression is often regulated by proteins, called transcription factors which bind to short (5 to 20 base pairs),degenerate segments of DNA. Experimental efforts towards understanding the sequence specificity of transcription factors is laborious and expensive, but can be substantially accelerated with the use of computational predictions. This thesis describes the use of algorithms and resources for transcriptionfactor binding site analysis in addressing quantitative modelling, where probabilitic models are built to represent binding properties of a transcription factor and can be used to find new functional binding sites in genomes. Initially, an open-access database(HTPSELEX) was created, holding high quality binding sequences for two eukaryotic families of transcription factors namely CTF/NF1 and LEFT/TCF. The binding sequences were elucidated using a recently described experimental procedure called HTP-SELEX, that allows generation of large number (> 1000) of binding sites using mass sequencing technology. For each HTP-SELEX experiments we also provide accurate primary experimental information about the protein material used, details of the wet lab protocol, an archive of sequencing trace files, and assembled clone sequences of binding sequences. The database also offers reasonably large SELEX libraries obtained with conventional low-throughput protocols.The database is available at http://wwwisrec.isb-sib.ch/htpselex/ and and ftp://ftp.isrec.isb-sib.ch/pub/databases/htpselex. The Expectation-Maximisation(EM) algorithm is one the frequently used methods to estimate probabilistic models to represent the sequence specificity of transcription factors. We present computer simulations in order to estimate the precision of EM estimated models as a function of data set parameters(like length of initial sequences, number of initial sequences, percentage of nonbinding sequences). We observed a remarkable robustness of the EM algorithm with regard to length of training sequences and the degree of contamination. The HTPSELEX database and the benchmarked results of the EM algorithm formed part of the foundation for the subsequent project, where a statistical framework called hidden Markov model has been developed to represent sequence specificity of the transcription factors CTF/NF1 and LEF1/TCF using the HTP-SELEX experiment data. The hidden Markov model framework is capable of both predicting and classifying CTF/NF1 and LEF1/TCF binding sites. A covariance analysis of the binding sites revealed non-independent base preferences at different nucleotide positions, providing insight into the binding mechanism. We next tested the LEF1/TCF model by computing binding scores for a set of LEF1/TCF binding sequences for which relative affinities were determined experimentally using non-linear regression. The predicted and experimentally determined binding affinities were in good correlation.

Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples

Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10-5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10-3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10-5, 10-4, and 10-3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 - 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 - 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 - 1.138)) laboratory results (Fig. 1-3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing.

Quantitative Ultrasound of the Heel and Fracture Risk Assessment: an Updated Meta-Analysis

Objectives: Quantitative ultrasound (QUS) is an attractive method for assessing fracture risk because it is portable, inexpensive, without ionizing radiation, and available in areas of the world where DXA is not readily accessible or affordable. However, the diversity of QUS scanners and variability of fracture outcomes measured in different studies is an important obstacle to widespread utilisation of QUS for fracture risk assessment. We aimed in this review to assess the predictive power of heel QUS for fractures considering different characteristics of the association (QUS parameters and fracture outcomes measured, QUS devices, study populations, and independence from DXA-measured bone density).Materials/Methods : We conducted an inverse-variance randomeffects meta-analysis of prospective studies with heel QUS measures at baseline and fracture outcomes in their follow-up. Relative risks (RR) per standard deviation (SD) of different QUS parameters (broadband ultrasound attenuation [BUA], speed of sound &SOS;, stiffness index &SI;, and quantitative ultrasound index [QUI]) for various fracture outcomes (hip, vertebral, any clinical, any osteoporotic, and major osteoporotic fractures) were reported based on study questions.Results : 21 studies including 55,164 women and 13,742 men were included with a total follow-up of 279,124 person-years. All four QUS parameters were associated with risk of different fractures. For instance, RR of hip fracture for 1 SD decrease of BUA was 1.69 (95% CI 1.43-2.00), SOS was 1.96 (95% CI 1.64-2.34), SI was 2.26 (95%CI 1.71-2.99), and QUI was 1.99 (95% CI 1.49-2.67). Validated devices from different manufacturers predicted fracture risks with a similar performance (meta-regression p-values>0.05 for difference of devices). There was no sign of publication bias among the studies. QUS measures predicted fracture with a similar performance in men and women. Meta-analysis of studies with QUS measures adjusted for hip DXA showed a significant and independent association with fracture risk (RR/SD for BUA =1.34 [95%CI 1.22-1.49]).Conclusions : This study confirms that QUS of the heel using validated devices predicts risk of different fracture outcomes in elderly men and women. Further research and international collaborations are needed for standardisation of QUS parameters across various manufacturers and inclusion of QUS in fracture risk assessment tools. Disclosure of Interest : None declared.

Quantitative morphometry analysis of the fetal brain using clinical MR imaging

In vivo fetal magnetic resonance imaging provides aunique approach for the study of early human braindevelopment [1]. In utero cerebral morphometry couldpotentially be used as a marker of the cerebralmaturation and help to distinguish between normal andabnormal development in ambiguous situations. However,this quantitative approach is a major challenge becauseof the movement of the fetus inside the amniotic cavity,the poor spatial resolution provided by very fast MRIsequences and the partial volume effect. Extensiveefforts are made to deal with the reconstruction ofhigh-resolution 3D fetal volumes based on severalacquisitions with lower resolution [2,3,4]. Frameworkswere developed for the segmentation of specific regionsof the fetal brain such as posterior fossa, brainstem orgerminal matrix [5,6], or for the entire brain tissue[7,8], applying the Expectation-Maximization MarkovRandom Field (EM-MRF) framework. However, many of theseprevious works focused on the young fetus (i.e. before 24weeks) and use anatomical atlas priors to segment thedifferent tissue or regions. As most of the gyraldevelopment takes place after the 24th week, acomprehensive and clinically meaningful study of thefetal brain should not dismiss the third trimester ofgestation. To cope with the rapidly changing appearanceof the developing brain, some authors proposed a dynamicatlas [8]. To our opinion, this approach however faces arisk of circularity: each brain will be analyzed /deformed using the template of its biological age,potentially biasing the effective developmental delay.Here, we expand our previous work [9] to proposepost-processing pipeline without prior that allow acomprehensive set of morphometric measurement devoted toclinical application. Data set & Methods: Prenatal MRimaging was performed with a 1-T system (GE MedicalSystems, Milwaukee) using single shot fast spin echo(ssFSE) sequences (TR 7000 ms, TE 180 ms, FOV 40 x 40 cm,slice thickness 5.4mm, in plane spatial resolution1.09mm). For each fetus, 6 axial volumes shifted by 1 mmwere acquired under motherâeuro?s sedation (about 1min pervolume). First, each volume is segmentedsemi-automatically using region-growing algorithms toextract fetal brain from surrounding maternal tissues.Inhomogeneity intensity correction [10] and linearintensity normalization are then performed. Brain tissues(CSF, GM and WM) are then segmented based on thelow-resolution volumes as presented in [9]. Ahigh-resolution image with isotropic voxel size of 1.09mm is created as proposed in [2] and using B-splines forthe scattered data interpolation [11]. Basal gangliasegmentation is performed using a levet setimplementation on the high-resolution volume [12]. Theresulting white matter image is then binarized and givenas an input in FreeSurfer software(http://surfer.nmr.mgh.harvard.edu) to providetopologically accurate three-dimensional reconstructionsof the fetal brain according to the local intensitygradient. References: [1] Guibaud, Prenatal Diagnosis29(4) (2009). [2] Rousseau, Acad. Rad. 13(9), 2006. [3]Jiang, IEEE TMI 2007. [4] Warfield IADB, MICCAI 2009. [5]Claude, IEEE Trans. Bio. Eng. 51(4) 2004. [6] Habas,MICCAI 2008. [7] Bertelsen, ISMRM 2009. [8] Habas,Neuroimage 53(2) 2010. [9] Bach Cuadra, IADB, MICCAI2009. [10] Styner, IEEE TMI 19(39 (2000). [11] Lee, IEEETrans. Visual. And Comp. Graph. 3(3), 1997. [12] BachCuadra, ISMRM 2010.

Quantitative multiparameter assays to measure the effect of adjuvants on human antigen-specific CD8 T-cell responses.

Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based vaccines have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic vaccines aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic vaccines to activate T cells, but more research is necessary to identify optimal compositions of potent vaccine formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigen-specific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during the last 15 years, and monitoring techniques become more and more standardized.

Prediction of hip fracture risk by quantitative ultrasound in more than 7000 Swiss women > or =70 years of age: comparison of three technologically different bone ultrasound devices in the SEMOF study.

To compare the prediction of hip fracture risk of several bone ultrasounds (QUS), 7062 Swiss women > or =70 years of age were measured with three QUSs (two of the heel, one of the phalanges). Heel QUSs were both predictive of hip fracture risk, whereas the phalanges QUS was not. INTRODUCTION: As the number of hip fracture is expected to increase during these next decades, it is important to develop strategies to detect subjects at risk. Quantitative bone ultrasound (QUS), an ionizing radiation-free method, which is transportable, could be interesting for this purpose. MATERIALS AND METHODS: The Swiss Evaluation of the Methods of Measurement of Osteoporotic Fracture Risk (SEMOF) study is a multicenter cohort study, which compared three QUSs for the assessment of hip fracture risk in a sample of 7609 elderly ambulatory women > or =70 years of age. Two QUSs measured the heel (Achilles+; GE-Lunar and Sahara; Hologic), and one measured the heel (DBM Sonic 1200; IGEA). The Cox proportional hazards regression was used to estimate the hazard of the first hip fracture, adjusted for age, BMI, and center, and the area under the ROC curves were calculated to compare the devices and their parameters. RESULTS: From the 7609 women who were included in the study, 7062 women 75.2 +/- 3.1 (SD) years of age were prospectively followed for 2.9 +/- 0.8 years. Eighty women reported a hip fracture. A decrease by 1 SD of the QUS variables corresponded to an increase of the hip fracture risk from 2.3 (95% CI, 1.7, 3.1) to 2.6 (95% CI, 1.9, 3.4) for the three variables of Achilles+ and from 2.2 (95% CI, 1.7, 3.0) to 2.4 (95% CI, 1.8, 3.2) for the three variables of Sahara. Risk gradients did not differ significantly among the variables of the two heel QUS devices. On the other hand, the phalanges QUS (DBM Sonic 1200) was not predictive of hip fracture risk, with an adjusted hazard risk of 1.2 (95% CI, 0.9, 1.5), even after reanalysis of the digitalized data and using different cut-off levels (1700 or 1570 m/s). CONCLUSIONS: In this elderly women population, heel QUS devices were both predictive of hip fracture risk, whereas the phalanges QUS device was not.

Evolutionary aspects of population structure for molecular and quantitative traits in the freshwater snail Radix balthica.

Detecting the action of selection in natural populations can be achieved using the QST-FST comparison that relies on the estimation of FST with neutral markers, and QST using quantitative traits potentially under selection. QST higher than FST suggests the action of directional selection and thus potential local adaptation. In this article, we apply the QST-FST comparison to four populations of the hermaphroditic freshwater snail Radix balthica located in a floodplain habitat. In contrast to most studies published so far, we did not detect evidence of directional selection for local optima for any of the traits we measured: QST calculated using three different methods was never higher than FST. A strong inbreeding depression was also detected, indicating that outcrossing is probably predominant over selfing in the studied populations. Our results suggest that in this floodplain habitat, local adaptation of R. balthica populations may be hindered by genetic drift, and possibly altered by uneven gene flow linked to flood frequency.