A novel wavelet-based thresholding method for the pre-processing of mass spectrometry data that accounts for heterogeneous noise

In recent years there has been an increased interest in using protein mass spectroscopy to discriminate diseased from healthy individuals with the aim of discovering molecular markers for disease. A crucial step before any statistical analysis is the pre-processing of the mass spectrometry data. Statistical results are typically strongly affected by the specific pre-processing techniques used. One important pre-processing step is the removal of chemical and instrumental noise from the mass spectra. Wavelet denoising techniques are a standard method for denoising. Existing techniques, however, do not accommodate errors that vary across the mass spectrum, but instead assume a homogeneous error structure. In this paper we propose a novel wavelet denoising approach that deals with heterogeneous errors by incorporating a variance change point detection method in the thresholding procedure. We study our method on real and simulated mass spectrometry data and show that it improves on performances of peak detection methods.     

A flow method for determination of the kinetic parameters for immobilized enzymes.

A flow method is described for determination of the kinetics parameters (V-m and K-m) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate of formation of product. To illustrate the technique, it is applied to the determination of the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing.     

A method for determining kinetic parameters at high enzyme concentrations.

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.     

A general population genetic framework for antagonistic selection that accounts for demography and recurrent mutation

Antagonistic selection--where alleles at a locus have opposing effects on male and female fitness ("sexual antagonism") or between components of fitness ("antagonistic pleiotropy")--might play an important role in maintaining population genetic variation and in driving phylogenetic and genomic patterns of sexual dimorphism and life-history evolution. While prior theory has thoroughly characterized the conditions necessary for antagonistic balancing selection to operate, we currently know little about the evolutionary interactions between antagonistic selection, recurrent mutation, and genetic drift, which should collectively shape empirical patterns of genetic variation. To fill this void, we developed and analyzed a series of population genetic models that simultaneously incorporate these processes. Our models identify two general properties of antagonistically selected loci. First, antagonistic selection inflates heterozygosity and fitness variance across a broad parameter range--a result that applies to alleles maintained by balancing selection and by recurrent mutation. Second, effective population size and genetic drift profoundly affect the statistical frequency distributions of antagonistically selected alleles. The "efficacy" of antagonistic selection (i.e., its tendency to dominate over genetic drift) is extremely weak relative to classical models, such as directional selection and overdominance. Alleles meeting traditional criteria for strong selection (N(e)s > 1, where N(e) is the effective population size, and s is a selection coefficient for a given sex or fitness component) may nevertheless evolve as if neutral. The effects of mutation and demography may generate population differences in overall levels of antagonistic fitness variation, as well as molecular population genetic signatures of balancing selection.     

A general method for constructing increment-decrement life tables that agree with the data

A general method for making increment-decrement life tables is presented. The method involves the finding of probabilities of transition between states, graduated to small intervals of time and age, that are consistent with (i.e., can reproduce) the data, whether the data consist of central age-state specific rates, or some other feature, such as state distributions of a real cohort. The method is then illustrated with a fetal loss life table.     

A general solution to the time interval omission problem applied to single channel analysis.

To obtain the open or closed time interval distributions of patch clamp signals, several workers have used a half-amplitude minimum time interval criterion. Within this framework, no transition between states of different conductance levels is considered to have taken place if it leads to a time interval smaller than a certain critical value. This procedure modifies substantially the open or closed time interval distribution of the random signal to be analyzed, since time intervals well above the time resolution of the recording system may be interrupted by short gaps that may or may not satisfy the minimum time interval criterion. We present here a general theoretical framework by means of which the effect of time interval omission on time interval distributions can be taken into account. Based on the mathematical formalism provided by the Kolmogorov forward equation, special matrix operators are first defined. The general solution to the time omission problem in its integral form is then derived. In view of the poor computational feasibility of the resulting solution, a first-order approximation is also presented. This approximation consists essentially in neglecting the contribution of the undetected gaps to the total length of the resulting time interval. The exact and approximate solutions are then applied to two special kinetic schemes commonly found in single-channel studies, namely the O-C and C-O-C models. The applicability of the proposed formalism to the time interval distribution problem of a damped random signal is finally discussed.     

A method for assessing alternative effects functions that uses simulation with EMDEX data

A method for evaluating a variety of alternative biologically plausible effects functions through the use of simulation studies conducted on personal-monitor exposure data is described. Using magnetic field time series collected with EMDEX instruments, we demonstrate how the method can be used to explore (1) how the outputs from various effects functions simulations compare to the results obtained by assuming that effects are proportional to time average field strength; (2) how the results of epidemiological studies might be used to assess the relative likelihood that each of the alternative effects functions describes biological reality; and (3) how the results might be used to assess possible health risks. Although the available data are sufficient to demonstrate the general method, they are not yet sufficient to support actual discrimination among possible alternatives. The arguments on the use of the method are for illustrative purposes only. © 1995 Wiley-Liss, Inc.     

A general method for studying the secretion of macromolecules by single cells: II. A simplified procedure with enhanced sensitivity

A simple, sensitive precipitin-type single-cell secretion assay is described and applied to the study of immunoglobulin-secreting cells. Evidence is presented that its efficiency is comparable to that achieved with reverse hemolytic plaque techniques. Also described is a modification of the simplified procedure which possesses substantially increased sensitivity. Enhanced sensitivity is achieved through the use of monoclonal rheumatoid factors which preferentially react with rabbit or human immunoglobulins that are incorporated into immune complexes as a result of interaction with antigen. Addition of rheumatoid factor to the agarose-cell mixture leads to additional crosslinking of immune complexes that form around active cells, thereby increasing the probability of forming a detectable precipitate. The application of this procedure to the detection of cells producing T-cell products is also discussed.     

Temperature dependence of kinetic parameters of single potential dependent K+ channels in mollusc neurons. Similarity in the action of potential and temperature]

Using the patch-voltage-clamp method on excised membrane fragments from molluscan neurones temperature dependences of kinetic parameters of the fast and slow K(+)-channels were investigated in the temperature range 1 to 40 degrees C. Temperature dependences of probability of the channel open state (P0) for the slow and fast K(+)-channels are, generally, opposite, that is P0 increases for the slow channel and decreases for the fast channel with temperature. Similar dependences characterize durations of single channel open intervals (tau 0) and burst durations (t(p)). Durations of interburst and interpulse intervals (respectively, t(i) and tau) decrease for the slow channel and increase, in contrast, for the fast channel with temperature. For the channels of both types temperature dependences of P0 (as for other parameters) are essentially nonmonotonous. There are two local extrema, at least: for the slow K(+)-channel-maximum at 15 degrees C (minimum for the fast channel) and minimum at 20-25 degrees C (maximum for the fast channel). In some cases the number of local extrema may be greater than two. Some similarity in the action of temperature and membrane potential on the kinetic parameters was observed. For the slow K(+)-channel P0, tau 0 and t p increase with temperature and membrane potential. For the fast channel these parameters decrease at the same conditions. Moreover, for the channels of both types temperature dependences of the kinetic parameters are slightly pronounced at the potentials where potential dependences of the parameters are least. As a whole, temperature measurements showed that there are, possibly, several points of structural transitions (similar to phase transitions) in the temperature range 0 to 40 degrees C. Primarily, the kinetic parameters are determined by these transitions.     

A semi-integrated method for the determination of enzyme kinetic parameters and graphical representation of the Michaelis-Menten equation

A semi-integrated method for the determination of the enzyme kinetics parameters (Km and V) and graphical representation of the Michaelis-Menten equation is proposed as a variation of determination of initial reaction rate (v) as a function of initial substrate concentration ([S]0). The method is based on the determination of the time required to exhaust half of the initial substrate concentration as a function of the initial substrate concentration. The advantages and limitations of this method are discussed.     

A general kinetic analysis of transport. Tests of the carrier model based on predicted relations among experimental parameters.

The analysis of transport kinetics has lacked both a unified treatment in which general rate equations are written entirely in terms of experimental parameters, and a convention by which these parameters may be designated in a concise yet immediately recognizable manner. Such a treatment is presented here in an easily accessible form, and a simple system of nomenclature is proposed resembling that in use in enzyme kinetics. The treatment is independent of assumptions about rate-limiting steps in transport, and applies to both active and facilitated systems, including obligatory exchange. A single substrate is characterized by twelve different parameters, only five of which are required in theory to calculate the others. If a second substrate is present on the trans side of the membrane there are six more parameters. All eighteen parameters are linked by multiple relationships which provide a complete set of rejection criteria for the generalized form of the mobile carrier. Relationships among parameters are also defined that give information on the rate-limiting steps in transport. Equations governing any individual experiment, involving only experimental parameters, are easily written out from the general expressions, for example under conditions of zero trans and infinite trans flux, equilibrium exchange, or competitive inhibition.     

A modification of the grain count halving method for detailed analysis of cell kinetic parameters

Abstract A modification of the conventional grain count halving (GCH) method is presented. By determining the decrease of the mean grain number of all interphase cells in addition to that of all labelled interphase cells on the same autoradiographs, the potential doubling time T _pot (or the cell production rate k _p) can be obtained in one and the same experiment. Thus the modified GCH method provides not only the cycle time of the cell population studied but also the growth fraction and, with additional cytofluorometric measurements, the duration of all cycle phases. By evaluating the cell production rate and the growth fraction this method leads to more reliable cell kinetic data of experimental tumours and human tumours growing in nude mice. In contrast to other cell kinetic methods, the modified GCH method can also be applied in special cases to human tumours in vivo , since only few points of measurement are needed. A comparison of the cell kinetic results obtained by the modified GCH method with those derived from the fraction of labelled mitoses method, both applied to allotransplants of the adenocarcinoma EO 771 in nude mice, shows good agreement.     

A modification of the grain count halving method for detailed analysis of cell kinetic parameters

A modification of the conventional grain count halving (GCH) method is presented. By determining the decrease of the mean grain number of all interphase cells in addition to that of all labelled interphase cells on the same autoradiographs, the potential doubling time Tpot (or the cell production rate kp) can be obtained in one and the same experiment. Thus the modified GCH method provides not only the cycle time of the cell population studied but also the growth fraction and, with additional cytofluorometric measurements, the duration of all cycle phases. By evaluating the cell production rate and the growth fraction this method leads to more reliable cell kinetic data of experimental tumours and human tumours growing in nude mice. In contrast to other cell kinetic methods, the modified GCH method can also be applied in special cases to human tumours in vivo, since only few points of measurement are needed. A comparison of the cell kinetic results obtained by the modified GCH method with those derived from the fraction of labelled mitoses method, both applied to allotransplants of the adenocarcinoma EO 771 in nude mice, shows good agreement.     

A general method for studying the secretion of macromolecules by single cells. I. Detection of immunoglobulin-secreting cells.

A general method for detecting single cells secreting macromolecules has been developed and applied to the detection of mouse cells secreting antibodies. Secreted Ig molecules were precipitated in the immediate vicinity of an active cell with rabbit anti-mouse Ig antibody. Rapid removal of excess antibody not incorporated into immunoprecipitates was achieved with an electrophoresis technique. The immuno-precipitate surrounding the active cell was then stained with sheep anti-rabbit Ig antibody labeled with horseradish peroxidase. The use of enzyme markers has made the procedure much more convenient and rapid than previous precipitin assays for cell secretion that used radioiodine for detecting immunoprecipitates. Moreover, the availability of several enzyme markers makes possible the detection of cells secreting more than one molecular species. Experiments were also run in which cells producing antibodies specific for horseradish peroxidase (HRP) could be identified among the population of cells producing other immunoglobulins. Presumably, HRP-labeled antigens could be used to identify cells producing other specific antibodies. The generality of this procedure suggests that it may be useful for detecting single T-cells releasing regulatory molecules, since specific antisera are already available for several of these molecules.     

A general method for the purification of restriction enzymes.

An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the sequencing, molecular cloning, and physical mapping of DNA.     

A general method for the lysis of Streptomyces species.

A procedure for the gentle lysis of Streptomyces is described. Complete lysis is obtained and covalently closed circular (CCC) DNA remains intact. This procedure was successfully used to lyse 14 different species of Streptomyces.