ROC graphs for assessing the ability of a diagnostic marker to detect three disease classes with an umbrella ordering

Receiver operating characteristic (ROC) curves and the area under these curves are commonly used to assess the ability of a continuous diagnostic marker (e.g., DNA methylation markers) to correctly classify subjects as having a particular disease or not (e.g., cancer). These approaches, however, are not applicable to settings where the gold standard yields more than two disease states or classes. ROC surfaces and the volume under the surfaces have been proposed for settings with more than two disease classes. These approaches, however, do not allow one to assess the ability of a marker to differentiate two disease classes from a third disease class without requiring a monotone order for the three disease classes under study. That is, existing approaches do not accommodate an umbrella ordering of disease classes. This article proposes the construction of an ROC graph that is applicable for an umbrella ordering. Furthermore, this article proposes that a summary measure for this umbrella ROC graph can be used to summarize the classification accuracy, and corresponding variance estimates can be obtained using U-statistics theory or bootstrap methods. The proposed methods are illustrated using data from a study assessing the ability of a DNA methylation marker to correctly classify lung specimens into three histologic classes: squamous cell carcinoma, large cell carcinoma, and nontumor lung.     

A comparison of nucleic acid amplification techniques for the assessment of bacterial viability

AIMS: The ability to determine the presence and viability status of bacteria by molecular methods could offer significant advantages to the food, environmental and health sectors, in terms of improved speed and sensitivity of detection. METHODS AND RESULTS: In this study, we have assessed three amplification techniques, PCR, RT-PCR and NASBA, for their ability to detect nucleic acid persistence in an E. coli strain following heat-killing. NASBA offered the greatest sensitivity of the three methods tested. The presence of residual DNA and mRNA could be detected by PCR and NASBA, respectively, for up to 30 h postdeath, by which time cell death had been confirmed by culture methods. Thus a single quantitative measurement based on nucleic acid amplification did not permit unequivocal determination of cell viability. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation between cell viability and persistence of nucleic acids must be well characterized for a particular analytical situation before molecular techniques can be substituted for traditional culture methods.     

Effect of air drying on bacterial viability: A multiparameter viability assessment

The effect of desiccation on the viability of microorganisms is a question of great interest for a variety of public health questions and industrial applications. Although viability is traditionally assessed by plate counts, cultivation-independent methods are increasingly applied with the aim to gain more insight into why cells might not form colonies and to optimize production processes. To evaluate their usefulness, we applied in this study a multiparameter viability assay to selected bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterococcus hirae, and Staphylococcus aureus) subjected to air-drying in the absence or presence of supplements. Tests included growth on solid culture medium and the measurement of membrane integrity, membrane potential, esterase and respiratory activities using fluorescent dyes. All measured parameters were responsive to desiccation stress. Results suggested that extending plate count analysis with cultivation-independent methods can greatly enhance resolution especially for moderate stress conditions, which do not get reflected in plate counts due to cellular recovery. Whereas plate counts reflect the final effect on viability, immediate measurement of cellular functions provides a snapshot picture of the fitness status at a specific point in time. Special emphasis was given to MgCl(2) which in concentrations≥50mM dramatically increased the bacterial susceptibility to desiccation in the case of the gram-negative bacteria and to a lesser extent also for the gram-positive bacteria. The study in addition confirmed a good agreement of results obtained with the recently developed real-time viability (RTV) assay and the BacLight LIVE/DEAD method in combination with a fluorescence plate reader.     

Flow cytometric assessment of cell viability: a multifaceted analysis

Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.     

Fecal pellet measurements of manipur brow-antlered deer with respect to sex and age classes

Measurements were made of fecal pellets for captive brow-antlered deer (Cervus eldi eldi) from different age and sex classes. Pellet measurements increased linearly with increases in age, except for length and width measurements in males. Differences in dimensions were statistically significant between sexes and among some age classes. This procedure may be useful in differentiating between the sexes, and to some extent between ages, of wild-living individuals. © 1993 Wiley-Liss, Inc.     

Quantitative assessment of the accuracy for three interpolation techniques in kinematic analysis of human movement

Marker obstruction during human movement analyses requires interpolation to reconstruct missing kinematic data. This investigation quantifies errors associated with three interpolation techniques and varying interpolated durations. Right ulnar styloid kinematics from 13 participants performing manual wheelchair ramp ascent were reconstructed using linear, cubic spline and local coordinate system (LCS) interpolation from 11-90% of one propulsive cycle. Elbow angles (flexion/extension and pronation/supination) were calculated using real and reconstructed kinematics. Reconstructed kinematics produced maximum elbow flexion/extension errors of 37.1 (linear), 23.4 (spline) and 9.3 (LCS) degrees. Reconstruction errors are unavoidable [minimum errors of 6.7?mm (LCS); 0.29?mm (spline); 0.42?mm (linear)], emphasising careful motion capture system setup must be performed to minimise data interpolation. For the observed movement, LCS-based interpolation (average error of 14.3?mm; correlation of 0.976 for elbow flexion/extension) was most suitable for reconstructing durations longer than 200?ms. Spline interpolation was superior for shorter durations.     

Quantitative assessment of the accuracy for three interpolation techniques in kinematic analysis of human movement

Marker obstruction during human movement analyses requires interpolation to reconstruct missing kinematic data. This investigation quantifies errors associated with three interpolation techniques and varying interpolated durations. Right ulnar styloid kinematics from 13 participants performing manual wheelchair ramp ascent were reconstructed using linear, cubic spline and local coordinate system (LCS) interpolation from 11–90% of one propulsive cycle. Elbow angles (flexion/extension and pronation/supination) were calculated using real and reconstructed kinematics. Reconstructed kinematics produced maximum elbow flexion/extension errors of 37.1 (linear), 23.4 (spline) and 9.3 (LCS) degrees. Reconstruction errors are unavoidable [minimum errors of 6.7 mm (LCS); 0.29 mm (spline); 0.42 mm (linear)], emphasising careful motion capture system setup must be performed to minimise data interpolation. For the observed movement, LCS-based interpolation (average error of 14.3 mm; correlation of 0.976 for elbow flexion/extension) was most suitable for reconstructing durations longer than 200 ms. Spline interpolation was superior for shorter durations.     

A comparative analysis of mutagenic and SOS-induced activity in three classes of chemical compounds

Mutagenic and SOS-inducing potential of 23 derivatives of fluorenone, phenanthrenequinone and biphenyl have been studied in tester strains of Salmonella typhimurium and in Escherichia coli strain PQ 37. 14 of these compounds revert the mutation hisD3052 (much less than -1 much greater than type), but none of them induce mutations in the strain TA 1535. Maximal mutagenic activity has been shown in strain TA 1538 for amide of 2,7-dinitrofluorenone-4-carbonic acid (580 revertants per nmol), 2,7-dinitrophenanthrenequinone (308 revertants per nmol), 2,4,7-trinitrophenanthrenequinone (306 revertants per nmol) and 2',4,4'-trinitrobiphenyl-2-carbonic acid (251 revertants per nmol). In plasmid-containing strain TA 98 the mutagenic potential of the compounds tested is lower than in the TA 1538 strain. It has been suggested that mutagenic activity of these compounds can be attributed to their acceptor properties, namely, the ability to form charge transfer complexes with DNA. SOS-inducing activity has been shown for 5 compounds, also positive in mutation induction. Mutagenic and SOS-inducing activities positively correlate in fluorenone derivatives. Among phenanthrenequinone derivatives, compounds with high mutagenic activity only can induce SOS response. None of the biphenyls tested induce SOS functions. The compounds giving the positive result in the SOS-chromotest have rigid co-planar structure.     

A global viability assessment of the European eel

The global European eel (Anguilla anguilla) stock is critically endangered according to the IUCN, and the European Commission has urged the development of conservation plans aimed to ensure its viability. However, the complex life cycle of this panmictic species, which reproduces in the open ocean but spends most of its prereproductive life in continental waters (thus embracing a huge geographic range and a variety of habitat types), makes it difficult to assess the long‐term effectiveness of conservation measures. The interplay between local and global stressors raises intriguing cross‐scale conservation challenges that require a comprehensive modelling approach to be addressed. We developed a full life cycle model of the global European eel stock, encompassing both the oceanic and the continental phases of eel's life, and explicitly allowing for spatial heterogeneity in vital rates, availability of suitable habitat and settlement potential via a metapopulation approach. We calibrated the model against a long‐term time series of global European eel catches and used it to hindcast the dynamics of the stock in the past and project it over the 21st century under different management scenarios. Although our analysis relies on a number of inevitable simplifying assumptions and on data that may not embrace the whole range of variation in population dynamics at the small spatiotemporal scale, our hindcast is consistent with the general pattern of decline of the stock over recent decades. The results of our projections suggest that (i) habitat loss played a major role in the European eel decline; (ii) the viability of the global stock is at risk if appropriate protection measures are not implemented; (iii) the recovery of spawner escapement requires that fishing mortality is significantly reduced; and (iv) the recovery of recruitment might not be feasible if reproductive output is not enhanced.     

Variation of lipid classes among organs of the Northern krill Meganyctiphanes norvegica, with respect to reproduction.

Lipid content and class in the Northern krill Meganyctiphanes norvegica (digestive gland, stomach, gonad, fat body, abdomen) was investigated and correlated with sex and reproductive stage. Ready to spawn females, have high lipid content in ovaries, while in males and spent females, the major site of lipid deposits was the digestive gland, followed by the fat body. These differences among spawning and spent females are indicative of strong interactions between the ovary and digestive gland and the ovary and fat body during vitellogenesis. Triacylglycerols (TAG) were the major neutral lipid class with high levels in the digestive gland. The major phospholipid was phosphatidylcholine (PC) particularly in the muscular tissue of the abdomen. Phosphatidyl-ethanolamine (PE) and -serine-inositol (PS-PI), were present at intermediate levels. Reproductive males were depleted in TAG and diacylglycerols (DAG) in the digestive gland, gonad and fat body, and had 4 times lower cholesterol in the gonad than ready to spawn females. Furthermore, ready to spawn females had in the ovary higher amounts of TAG, DAG and phospholipids (PC, PE, PS-PI) than spent females. Linear relationships between lipid content and main lipid class (TAG, PC, PE, PS-PI) in different fractions of males and ready to spawn females showed that: (1). TAG was stored for both sexes in all cephalothorax fractions with highest values in the digestive gland and ovary fluid; (2). PC was accumulated for both sexes in the fat body and the gonad with a higher slope for females, with the highest values in the ovary fluid and in the abdomen of males and that (3). PS-PI was stored only in the ovary and abdomen of mature females. These results are discussed in terms of the strategy developed by Meganyctiphanes norvegica to allocate lipids to the next generation for optimised embryogenesis.     

Ecological features of migrating passerines (analysis with molecular biology techniques involved)

The adaptation mechanisms in Phylloscopus warblers to habitat conditions associated with their reproduction, namely, the sex ratio, family structure, duration of sexual activity, etc., have been studied. The brood sex ratio is established to be about 1: 1 among juveniles, but males begin to predominate with time. In the reproductive period, some males remain unmated for a long time or even over the whole season. On the other hand, nesting males create pairs with second females. Visual observations and the results of a family structure analysis by means of molecular-genetic methods have shown that female “adultery” and/or forcible copulations with alien males occur from time to time, which leads to the appearance of extra-pair paternity. A PCR analysis revealed a high level of genetic diversity within the local populations of all the Phylloscopus warbler species, but the diversity for wood and greenish warblers (new species for the region) was lower than that for chiff-chaffs and willow warblers (species native to the region).     

Morphological characterization and viability assessment of Trichoderma reesei by image analysis

The production of cellulase from the filamentous fungus Trichoderma reesei is a critical step in the industrial process leading to cellulose ethanol. As a result of the lack of quantitative analysis tools, the intimate relationship that exists between the morphological and physiological states of the microorganism, the shear field in the bioreactor, and the process performance is not yet fully understood. A semiautomatic image analysis protocol was developed to characterize the mycelium morphology and to estimate its percentage viability during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms). Pictures taken under bright field microscopy combined with images of fluorescein diacetate stained fungi were used to assess the morphological parameters and the percentage viability of microorganisms simultaneously. The method was tested during the course of fed-batch fermentation in a reciprocating plate bioreactor. The use of the image analysis protocol was found to be successful in quantifying the variations in the morphology and the viability of T. reesei throughout the fermentation.     

Comparison of three methods for beat-to-beat-interval extraction from continuous blood pressure and electrocardiogram with respect to heart rate variability analysis.

In recent years the analysis of heart rate variability (HRV) has become a suitable method for characterizing autonomous cardiovascular regulation. The aim of this study was to investigate the differences in HRV estimated from continuous blood pressure (BP) measurement by different methods in comparison to electrocardiogram (ECG) signals. The beat-to-beat intervals (BBI) were simultaneously extracted from the ECG and blood pressure of 9 cardiac patients (10 min, Colin system, 1000-Hz sampling frequency). For both data types, slope, peak, and correlation detection algorithms were applied. The short-term variability was calculated using concurrent 10-min BP and ECG segments. The root mean square errors in comparison to ECG slope detection were: 1.74 ms for ECG correlation detection; 5.42 ms for ECG peak detection; 5.45 ms for BP slope detection; 5.75 ms for BP correlation detection; and 11.96 ms for BP peak detection. Our results show that the variability obtained with ECG is the most reliable. Moreover, slope detection is superior to peak detection and slightly superior to correlation detection. In particular, for ECG signals with higher frequency characteristics, peak detection often exhibits more artificial variability. Besides measurement noise, respiratory modulation and pulse transit time play an important role in determining BBI. The slope detection method applied to ECG should be preferred, because it is more robust as regards morphological changes in the signals, as well as physiological properties. As the ECG is not recorded in most animal studies, distal pulse wave measurement in combination with correlation or slope detection may be considered an acceptable alternative.     

A comparative analysis of various developmental stages of Schistosoma mansoni with respect to their protein composition

The protein composition of Schistosoma mansoni was analysed by electrophoresis in polyacrylamide slab gel in the presence of sodium dodecyl sulphate. A high degree of identity existed between protein patterns obtain from immature and adult parasites of the two sexes. Some stage-and sex-specific components were indentified and their possible origin discussed.     

Phytoextraction: an assessment of biogeochemical and economic viability

Phytoextraction describes the use of plants to remove metals and other contaminants from soils. This low-cost technology has potential for the in situ remediation of large areas of contaminated land. Despite more than 10 years of intensive research on the subject, very few commercial phytoextraction operations have been realised. Here, we investigate the viability of phytoextraction as an effective land-treatment technology. A Decision Support System (DSS) was developed to predict the effect of phytoextraction on soil metal concentration and distribution, as well as the economic feasibility of the process in comparison to either inaction or the best alternative technology. Changes in soil metal concentration are mechanistically predicted on the basis of plant water use, metal concentration in soil solution, soil density, plant root distribution and our so-called root-absorption factor. The root-absorption factor is a `lumped parameter' describing the xylem/soil solution metal concentration quotient. Phytoextraction is considered to be a viable option if it can satisfy environmental regulations and simultaneously be shown to be the most cost-effective technology, either alone, or in combination with other remediation technologies. To date, commercial phytoextraction has been constrained by the expectation that site remediation should be achieved in a time comparable to other clean-up technologies. However, if phytoextraction could be combined with a profit making operation such as forestry, then this time constraint, which has often been considered to be the Achilles heel of phytoextraction, may be less important.     

Interethnic comparisons of three endogamous groups of West Bengal with respect to two sets of variables

The present paper focuses on the interethnic comparison among three endogamous groups (a higher caste, a tribe and a religious group) with different ethnohistorical background. A total of 700 individuals were studied from their anthropometric and dermatoglyphic traits along with their asymmetries. Statistical comparisons were carried out through the use of student’s ‘t’ test and Sanghvi’s T² statistics. Heterogeneity for most of the anthropometric and some dermatoglyphic traits supports their different origins. Though dermatoglyphic asymmetry doesn’t reveal significant discrimination, heterogeneity in anthropometric asymmetry corroborates with their different occupations. The anthropometric distances show greater values than the dermatoglyphic distances. This difference is probably due to the fact that anthropometry is not independent of environment and not a stable character like dermatoglyphic traits.     

Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation

A flexible statistical framework is developed for the analysis of read counts from RNA-Seq gene expression studies. It provides the ability to analyse complex experiments involving multiple treatment conditions and blocking variables while still taking full account of biological variation. Biological variation between RNA samples is estimated separately from the technical variation associated with sequencing technologies. Novel empirical Bayes methods allow each gene to have its own specific variability, even when there are relatively few biological replicates from which to estimate such variability. The pipeline is implemented in the edgeR package of the Bioconductor project. A case study analysis of carcinoma data demonstrates the ability of generalized linear model methods (GLMs) to detect differential expression in a paired design, and even to detect tumour-specific expression changes. The case study demonstrates the need to allow for gene-specific variability, rather than assuming a common dispersion across genes or a fixed relationship between abundance and variability. Genewise dispersions de-prioritize genes with inconsistent results and allow the main analysis to focus on changes that are consistent between biological replicates. Parallel computational approaches are developed to make non-linear model fitting faster and more reliable, making the application of GLMs to genomic data more convenient and practical. Simulations demonstrate the ability of adjusted profile likelihood estimators to return accurate estimators of biological variability in complex situations. When variation is gene-specific, empirical Bayes estimators provide an advantageous compromise between the extremes of assuming common dispersion or separate genewise dispersion. The methods developed here can also be applied to count data arising from DNA-Seq applications, including ChIP-Seq for epigenetic marks and DNA methylation analyses.     

The effects of biogenic and abiogenic disulphides on endothelial cells in culture: Comparison of three methods of viability assessment

Effects of biogenic and abiogenic disulfides on viability of human umbilical vein endothelial cells in culture were studied using three methods: inclusion of neutral red (NR) into cells, quantification of intracellular ATP, and modifications of the Mosmann method (1983), the essence of which consists in reduction of tetrazolim salts—MTT and MTS—by cells (Mosmann, 1983). 2,2′-Dithio-bis(N,N-diethyl)dietanamine (DS) was used as an abiogenic sulfide. As for biogenic disulfides, we used oxydized glutathione (GSSG) and garlic oil (GO), the major component of which is diallyl disulfide (DADS). It has been established that DS and GO have a compatible cytotoxic effect (ES₅₀ amounts to ～0.6 mM), whereas GSSG at a concentration up to 1 mM had no effect on the viability of endothelial cells. It has been demonstrated for the first time that DS and GO can serve as mediators of plasma-membrane oxidoreductase activity, with tetrazolium salts being used as the substrate, which may cause a false-negative effect. In this connection, Mosmann’s method has serious limitations in evaluating the effect of disulfides on cell viability, although it can be used when studying the mechanism of their action.