A microcomputer program and fast analog to digital converter card for the analysis of fluorescence induction transients

A microcomputer program and an analog to digital conversion card were developed for the analysis of fluorescence induction curves. The program and the analog to digital conversion card are compatible with all commercially available fluorometers. Most of the current analysis methods for fluorescence induction curves are implemented in the program, including analysis of OIDPSMT-kinetics, dissection of fluorescence quenching into two components, measurement of the slope of the fluorescence curve, complementary area analysis and analysis of energy spillover from PS II to PS I. The program can also do basic statistical calculations from the measured parameters. The architecture of the program is open, allowing the user to add new methods to the main body of the program. Split time-scale is used in data capture and analysis. A new procedure facilitates accurate determination of F₀.     

Determination of the antenna heterogeneity of Photosystem II by direct simultaneous fitting of several fluorescence rise curves measured with DCMU at different light intensities

Several methods for determination of the antenna heterogeneity of Photosystem II from fluorescence rise curves measured with DCMU have been developed so far. Using these methods, two, three or four types of Photosystem II with respect to the antenna heterogeneity were determined. However, the accuracy of some of these methods is under debate. Here, we present a new method for the determination of the antenna heterogeneity of Photosystem II. The method is based on direct simultaneous fitting of several fluorescence rise curves measured with DCMU at different intensities of light excitation. As several curves measured under different light conditions are fitted simultaneously by the same model, reliability and accuracy in determination of model parameters increase. Our method was applied to two plant materials with different structure of the thylakoid membrane: wheat leaves and cells of green alga Chlamydomonas reinhardtii. This revised version was published online in June 2006 with corrections to the Cover Date.     

A New Testing Approach for Comparing the Overall Homogeneity of Survival Curves

The assessment of overall homogeneity of time‐to‐event curves is a key element in survival analysis. The currently commonly used methods, e.g., log‐rank and Wilcoxon tests, may have a significant loss of statistical testing power under certain circumstances. In this paper a new statistical testing approach is developed to compare the overall homogeneity of survival curves. The proposed new method has greater power than the commonly used tests to detect overall differences between crossing survival curves. The small‐sample performance of the new test is investigated under a variety of situations by means of Monte Carlo simulations. Furthermore, the applicability of the proposed testing approach is illustrated by a real data example from a kidney dialysis trial. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Dual fluorophore-nitroxide probes for analysis of vitamin C in biological liquids

A new method for quantitative analysis of vitamin C in biological and chemical liquids was proposed. The method is based on the use of dual molecule consisting of a fluorescent chromophore and a nitroxide radical. In the dual molecule, the nitroxide acts as a quencher of the fluorescence of the chromophore fragment. Reduction of the nitroxide fragment by ascorbic acid results in decay of ESR signal and enhancement of the fluorescence. By performing the series of pseudo-first-order reactions between the dual molecule and ascorbic acid and consequent plotting rate constants versus ascorbic acid concentrations the calibration curves for the vitamin C analysis were obtained. Variations of chemical structure of fluorophore and nitroxide fragments allow to regulate fluorescent properties and redox potentials of the dual molecules. The proposed fluorophore-nitroxide hybrids retain all features of the spin labels and fluorescence probes gaining new advantages for monitoring redox reactions and radical processes by two independent techniques: ESR and steady-state fluorescent spectroscopy. The method was applied to the vitamin C analysis in commercial fruit juices.     

Tilted, extended, and lying in wait: the membrane-bound topology of residues Lys-381-Ser-405 of the colicin E1 channel domain

The membrane-bound closed state (zero potential) of the helix 3 segment (Lys-381-Ser-405) of the colicin E1 channel domain was investigated by site-directed fluorescence labeling using a bimane probe tethered to a single cysteine residue of each mutant protein. A number of fluorescence properties of the tethered bimane probe were measured for the soluble channel mutant proteins as well as for the membrane-bound proteins. A new method called helical periodicity surface analysis was employed to fit the fluorescence data to a harmonic wave function using four different statistical methods. The fit of the various data sets to a harmonic wave function indicated that the periodicity of helix 3 in the membrane-bound state is typical for an amphipathic alpha helix (3.7-4.0 residues per turn and an angular frequency between 90 and 97 degrees). Notably, upon membrane binding, helix 3 elongates from 15 residues (soluble structure) to 20 residues by a three- and two-residue extension at the N- and C-termini of the helix, respectively. Dual quencher analysis also revealed that helix 3 is appressed to the surface of the membrane with its N-terminus more deeply buried within the interfacial region of the bilayer than its C-terminus. Finally, contrary to a previous report, our data show that helices 3 and 4 remain separate and independent helices upon membrane association in the absence of a membrane potential.     

A new method for measuring passive length-tension properties of human gastrocnemius muscle in vivo

The study of muscle growth and muscle length adaptations requires measurement of passive length-tension properties of individual muscles, but until now such measurements have only been made in animal muscles. We describe a new method for measuring passive length-tension properties of human gastrocnemius muscles in vivo. Passive ankle torque and ankle angle data were obtained as the ankle was rotated through its full range with the knee in a range of positions. To extract gastrocnemius passive length-tension curves from passive torque-angle data it was assumed that passive ankle torque was the sum of torque due to structures which crossed only the ankle joint (this torque was a 6-parameter function of ankle joint angle) and a torque due to the gastrocnemius muscle (a 3-parameter function of knee and ankle angle). Parameter values were estimated with non-linear regression and used to reconstruct passive length-tension curves of the gastrocnemius. The reliability of the method was examined in 11 subjects by comparing three sets of measurements: two on the same day and the other at least a week later. Length-tension curves were reproducible: the average root mean square error was 5.1+/-1.1 N for pairs of measurements taken within a day and 7.3+/-1.2 N for pairs of measurements taken at least a week apart (about 3% and 6% of maximal passive tension, respectively). Length-tension curves were sensitive to mis-specification of moment arms, but changes in length-tension curves were not. The new method enables reliable measurement of passive length-tension properties of human gastrocnemius in vivo, and is likely to be useful for investigation of changes in length-tension curves over time.     

ConvAn: a convergence analyzing tool for optimization of biochemical networks

Dynamic models of biochemical networks usually are described as a system of nonlinear differential equations. In case of optimization of models for purpose of parameter estimation or design of new properties mainly numerical methods are used. That causes problems of optimization predictability as most of numerical optimization methods have stochastic properties and the convergence of the objective function to the global optimum is hardly predictable. Determination of suitable optimization method and necessary duration of optimization becomes critical in case of evaluation of high number of combinations of adjustable parameters or in case of large dynamic models. This task is complex due to variety of optimization methods, software tools and nonlinearity features of models in different parameter spaces. A software tool ConvAn is developed to analyze statistical properties of convergence dynamics for optimization runs with particular optimization method, model, software tool, set of optimization method parameters and number of adjustable parameters of the model. The convergence curves can be normalized automatically to enable comparison of different methods and models in the same scale. By the help of the biochemistry adapted graphical user interface of ConvAn it is possible to compare different optimization methods in terms of ability to find the global optima or values close to that as well as the necessary computational time to reach them. It is possible to estimate the optimization performance for different number of adjustable parameters. The functionality of ConvAn enables statistical assessment of necessary optimization time depending on the necessary optimization accuracy. Optimization methods, which are not suitable for a particular optimization task, can be rejected if they have poor repeatability or convergence properties. The software ConvAn is freely available on www.biosystems.lv/convan.     

A model for thermograms

Thermograms are curves resulting from thermal analysis and are of great interest in the study of various food and biological products physical properties. A method to separate underlying peaks is proposed, and statistical properties of estimates for some characteristic parameters are derived. The total number of peaks can be estimated with a sequential analysis of the residual plots. For each new peak, a statistical criterion is proposed to check whether it is significantly different from the noise of the recording. As an example, the method is applied to a summer milk fat fusion thermogram.     

Ligand binding systems at equilibrium: specificity, heterogeneity, cross-reactivity, and site-site interactions

The simplest ligand binding model that can account for systems which consist of heterogeneous receptors that show site-site interactions and can react with several types of ligands was presented. This model was based on the grand canonical partition function whose properties and potentialities for obtaining binding functions are briefly illustrated. A computer simulation of this model was carried out in order to examine the contributions of site-site interactions on the shape of binding isotherms and on specificity curves. The shape of the binding isotherms was shown to be independent regardless of whether the path of binding was considered. However, the shape of the specificity curves was strongly dependent on site-site interactions and on the particular sequence of ligand-receptor configurations formed during the ligand binding process, leading to the conclusion that site-site interactions may influence the specificity of a system more than even very strong cross-reactions. A method based on the Ising model of statistical mechanics was also described and was used for obtaining binding functions applicable to infinite lattices which show site-site interactions and competitive binding. The results presented here point out possible errors that can arise if standard statistical methods are used to fit binding data in order to prove a given binding model.     

Stochastic dynamic study of optical transition properties of single GFP-like molecules

Due to high fluctuations and quantum uncertainty, the processes of single-molecules should be treated by stochastic methods. To study fluorescence time series and their statistical properties, we have applied two stochastic methods, one of which is an analytic method to study the off-time distributions of certain fluorescence transitions and the other is Gillespie’s method of stochastic simulations. These methods have been applied to study the optical transition properties of two single-molecule systems, GFPmut2 and a Dronpa-like molecule, to yield results in approximate agreement with experimental observations on these systems. Rigorous oscillatory time series of GFPmut2 before it unfolds in the presence of denaturants have not been obtained based on the stochastic method used, but, on the other hand, the stochastic treatment puts constraints on the conditions under which such oscillatory behavior is possible. Furthermore, a sensitivity analysis is carried out on GFPmut2 to assess the effects of transition rates on the observables, such as fluorescence intensities.     

Further fluorospectrophotometric studies on the binding of acridine orange with DNA. Effects of thermal denaturation of DNA and additions of spermine, kanamycin, dihydrostreptomycin, methylene blue and chlorpromazine

Fluorospectrophotometric studies on the binding of acridine orange (AO) with calf thymus DNA showed that the thermal denaturation of DNA reduced markedly the fluorescence of Complex II and the extent of this decrease depended on the temperature to which the DNA solutions were heated. The denaturation was carried out in the absence and presence of AO (methods A and B, respectively), and then fluorescence measurements of solutions were carried out at 23 °C. The fluorescence intensity-heating temperature curves obtained by methods A and B were similar in shape to the usual melting curves of DNA and AO-DNA solutions, respectively. The higher midpoint value obtained with method B indicates the stabilizing activity of AO against denaturation. These findings support an intercalation model for Complex II and an external self-association binding model for Complex I.A high concentration of ethylene diamine (EDA) restored the fluorescence of denatured Complex II to about 80% of the intensity value of native Complex II. The effects of spermine, kanamycin and dihydrostreptomycin were much stronger than that of EDA.Methylene blue (MB) and chlorpromazine (CP) reduced the fluorescence of native Complex II markedly. Since the analysis of the difference absorption spectra declared that MB and CP were intercalated without release of bound AO, the interacting MB and CP were considered to weaken the interaction between AO and DNA bases, that made AO more fluorescent. Free radical (CP·) of CP was prepared by a new method using H₂O₂, peroxidase, and ascorbic acid. Intercalated CP· showed a much stronger quenching effect on Complex II, indicating that unpaired electron spin contained in the costacking unit between CP· and DNA bases might affect the fluorescence of the adjacent AO molecule by paramagnetic perturbation.     

Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein-Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.     

Estimation of the Transition/Transversion Ratio

A simple method for estimating the transition/transversion ratio was developed. This method can be applied to not only two sequences but also more than two sequences. The statistical properties of the method and some other methods were examined by numerical computation and computer simulation. The results obtained showed that, in terms of bias and variance, the new method gives a better estimate of the transition/transversion ratio than do the other examined methods. The new method was applied to human and chimpanzee mitochondrial control region sequences. Received: 22 September 1997 / Accepted: 1 November 1997     

Revisiting the sigmoidal curve fitting applied to quantitative real-time PCR data

Amplification of a cDNA product by quantitative polymerase chain reaction (qPCR) gives rise to fluorescence sigmoidal curves from which absolute or relative target gene content of the sample is inferred. Besides comparative C(t) methods that require the construction of a reference standard curve, other methods that focus on the analysis of the sole amplification curve have been proposed more recently. Among them, the so-called sigmoidal curve fitting (SCF) method rests on the fitting of an empirical sigmoidal model to the experimental amplification data points, leading to the prediction of the amplification efficiency and to the calculation of the initial copy number in the sample. The implicit assumption of this method is that the sigmoidal model may describe an amplification curve quantitatively even in the portion of the curve where the fluorescence signal is hidden in the noise band. The theoretical basis of the SCF method was revisited here for defining the class of experimental amplification curves for which the method might be relevant. Applying the SCF method to six well-characterized different PCR assays illustrated possible pitfalls leading to biased estimates of the amplification efficiency and, thus, of the target gene content of a sample.     

A New Distribution-Free Approach to Constructing the Confidence Region for Multiple Parameters

Construction of confidence intervals or regions is an important part of statistical inference. The usual approach to constructing a confidence interval for a single parameter or confidence region for two or more parameters requires that the distribution of estimated parameters is known or can be assumed. In reality, the sampling distributions of parameters of biological importance are often unknown or difficult to be characterized. Distribution-free nonparametric resampling methods such as bootstrapping and permutation have been widely used to construct the confidence interval for a single parameter. There are also several parametric (ellipse) and nonparametric (convex hull peeling, bagplot and HPDregionplot) methods available for constructing confidence regions for two or more parameters. However, these methods have some key deficiencies including biased estimation of the true coverage rate, failure to account for the shape of the distribution inherent in the data and difficulty to implement. The purpose of this paper is to develop a new distribution-free method for constructing the confidence region that is based only on a few basic geometrical principles and accounts for the actual shape of the distribution inherent in the real data. The new method is implemented in an R package, distfree.cr/R. The statistical properties of the new method are evaluated and compared with those of the other methods through Monte Carlo simulation. Our new method outperforms the other methods regardless of whether the samples are taken from normal or non-normal bivariate distributions. In addition, the superiority of our method is consistent across different sample sizes and different levels of correlation between the two variables. We also analyze three biological data sets to illustrate the use of our new method for genomics and other biological researches.     

A new approach to the detection and statistical classification of Ca2+ sparks

The availability of high-speed, two-dimensional (2-D) confocal microscopes and the expanding armamentarium of fluorescent probes presents unprecedented opportunities and new challenges for studying the spatial and temporal dynamics of cellular processes. The need to remove subjectivity from the detection process, the difficulty of the human eye to detect subtle changes in fluorescence in these 2-D images, and the large volume of data produced by these confocal microscopes call for the need to develop algorithms to automatically mark the changes in fluorescence. These fluorescence signal changes are often subtle, so the statistical estimate of the likelihood that the detected signal is not noise is an integral part of the detection algorithm. This statistical estimation is fundamental to our new approach to detection; in earlier Ca(2+) spark detectors, this statistical assessment was incidental to detection. Importantly, the use of the statistical properties of the signal local to the spark, instead of over the whole image, reduces the false positive and false negative rates. We developed an automatic spark detection algorithm based on these principles and used it to detect sparks on an inhomogeneous background of transverse tubule-labeled rat ventricular cells. Because of the large region of the cell surveyed by the confocal microscope, we can detect a large enough number of sparks to measure the dynamic changes in spark frequency in individual cells. We also found, in contrast to earlier results, that cardiac sparks are spatially symmetric. This new approach puts the detection of fluorescent signals on a firm statistical foundation.     

Comparison of different approaches for comparative genetic analysis using microarray hybridization

A robust analysis of comparative genomic microarray data is critical for meaningful genomic comparison studies. In this paper, we compare our method (implemented in a new software tool, GENCOM, freely available at ) with three commonly used analysis methods: GACK (freely available at ), an empirical cut-off value of twofold difference between the fluorescence intensities after LOWESS normalization or after AVERAGE normalization in which the fluorescence intensity is divided by the average fluorescence intensity of the entire data set. Each method was tested using data sets from real experiments with prior knowledge of conserved and divergent genes. GENCOM and GACK were superior when a high proportion of genes were divergent. GENCOM was the most suitable method for the data set in which the relationship between the fluorescence intensities was not linear. GENCOM has proved robust in an analysis of all the data sets tested.     

Context based mixture model for cell phase identification in automated fluorescence microscopy

Background  

Automated identification of cell cycle phases of individual live cells in a large population captured via automated fluorescence microscopy technique is important for cancer drug discovery and cell cycle studies. Time-lapse fluorescence microscopy images provide an important method to study the cell cycle process under different conditions of perturbation. Existing methods are limited in dealing with such time-lapse data sets while manual analysis is not feasible. This paper presents statistical data analysis and statistical pattern recognition to perform this task.     

Characterization of Photosynthetic Rhythms in Marine Dinoflagellates: II. Photosynthesis-Irradiance Curves and in Vivo Chlorophyll a Fluorescence

Using data from light-dark cultures of Gonyaulax polyedra entrained to a 24-hour cycle, whole cell absorption curves and photosynthesis-irradiance curves were constructed for various circadian times. While whole cell absorbance and half-saturation constants of photosynthesis showed no statistical difference that could be directly related to the photosynthetic rhythm, the initial slope of the photosynthesis-irradiance curve was a time-dependent parameter which altered in direct proportion to the change in photosynthetic capacity. The results indicated a temporal change in the relative quantum yield of photosynthesis, and the circadian rhythmicity of light-limited photosynthesis was established under constant conditions. Circadian rhythmicity was detected in room temperature chlorophyll fluorescence yield. Low temperature fluorescence kinetics also showed fluctuations. The results suggest that regulation of photosynthesis by the biological clock of Gonyaulax may be mediated through the membrane-bound light reactions and a partial explanation of the underlying mechanism is proposed.     

Stopped-flow solution scattering using synchrotron radiation: Apparatus,data collection and data analysis

We have constructed an experimental system, under remote control, for stopped-flow X-ray scattering using synchrotron radiation. It has been used, in conjunction with an annular detector and its associated electronics, to obtain good scattering curves, with time-slices as short as 200 ms, in a new study of the dissociation of the enzyme complex aspartate transcarbamylase. The data have been analysed by new statistical methods, and they agree well with the results from parallel chemical quench experiments. For studying dissociation reactions, stopped-flow X-ray scattering is a quite practical method, which need not use very much more material than conventional stopped-flow experiments.