A two-state recurrent stochastic model with time-dependent transition rates.

The two-state recurrent stochastic model with time-independent transition rates is generalized to a model with time-dependent transition rates. The rates can be any general function of external time, that is, any general function of the calendar time in which the process unfolds. Formulas for the state transition probabilities, the proportion of individuals in a particular state at time t, the distribution function, and the expectation of the number of individuals in a particular state at time t are derived.     

The statistical dynamics of recurrent biological events

In this paper we develop a general modeling framework within which many models for systems which produce events at irregular times through a combination of probabilistic and deterministic dynamics can be comprehended. We state and prove new sufficient conditions for the global asymptotic behaviour of the density evolution in these systems, and apply our results to many previously published models for the cell division cycle. In addition, we develop a new interpretation for the statistics of action potential production in excitable cells.To whom correspondence should be sent     

Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity.

BACKGROUND:Current cytotoxic assays, including Cr release and fluorescent assays, do not directly measure the proportion of target cells which are killed by apoptosis. Cell-mediated cytotoxicity induced by CTLs and NK cells is mainly regulated by the perforin-granzyme, the Fas ligand (Fas L), and the Tumor Necrosis Factor (TNF)-alpha pathways. Perforin generates pores in the membrane of target cells, allowing granzyme B to enter and initiate apoptosis. The other effectors, Fas L and TNF-alpha act by an apoptosis mechanism, leading to DNA fragmentation. A three color flow cytometric method to measure cell-mediated cytotoxicity induced by CTLs or NK cells is described. METHODS:The fluorochromes used are: PKH-26, a stable membrane dye for the labeling of the effector cells, annexin V-FITC which allows the direct evaluation of early apoptotic cells and propidium iodide which distinguishes membrane permeabilized and late apoptotic cells. RESULTS:By eliminating through gating PKH-26 positive effector cells, we obtain a direct estimation of the percentage of target cells in the early stages of apoptosis as well as the percentage of target cells dying after late apoptosis and membrane permeabilization. The cytotoxic activity of IL-2 stimulated PBL against K562, Jurkat and KYM-1 was evaluated. CONCLUSIONS:This rapid and novel assay permits the discrimination of the cell death mechanisms occurring during a cytotoxic response and to precisely evaluate the contribution of apoptosis in the early phases of cell-mediated cytotoxicity.     

Amino acid flooding doses for measuring rates of protein synthesis

Summary The development, advantages and disadvantages of using the amino acid flooding dose technique to determinein vivo rates of protein synthesis are examined in this review. A discussion of the use of this procedure in animals greater than 5 kg is included. The flooding dose procedure reduces the disparity between isotope enrichment in different amino acid precursor pools, which should theoretically improve the precision and accuracy of protein synthesis measurements. However, the possibility must be considered that the large doses of amino acids injected or infused in conjunction with this technique may influence protein turnover due to attendant metabolic effects. Therefore, a judicious choice of an amino acid and an evaluation of the experimental parameters involved in this procedure are required to optimize the accuracy of results obtained.Scientific Paper No. 710. Agriculture Canada, Lacombe Research Station, Bag Service 5000, Lacombe, Alberta T0C 1S0.     

Characterization of cycA mutants of Escherichia coli. An assay for measuring in vivo mutation rates

Quantitative assessment of the spontaneous or induced genomic mutation rate, a fundamental evolutionary parameter, usually requires the use of well-characterized mutant selection systems. Although there is a great number of genetic selection schemes available in Escherichia coli, the selection of D-cycloserine resistant mutants is shown here to be particularly useful to yield a general view of mutation rates and spectra. The combination of a well-defined experimental protocol with the Ma-Sandri-Sarkar maximum likelihood method of fluctuation analysis results in reproducible data, adequate for statistical comparisons. The straightforward procedure is based on a simple phenotype-genotype relationship, and detects mutations in the single-copy, chromosomal cycA gene, involved in the uptake of D-cycloserine. In contrast to the widely used rifampicin resistance assay, the procedure selects mutations which are neutral in respect of cell growth. No specific genetic background is needed, and practically the entire mutation spectrum (base substitutions, frameshifts, deletions, insertions) can simultaneously be measured. A systematic analysis of cycA mutations revealed a spontaneous mutation rate of 6.54 x 10(-8) in E. coli K-12 MG1655. The mutation spectrum was dominated by point mutations (base substitutions, frameshifts), spread over the entire gene. IS insertions, caused by IS1, IS2, IS3, IS4, IS5 and IS150, represented 24% of the mutations.     

Identification of recurrent regulated alternative splicing events across human solid tumors

Cancer is a complex disease that involves aberrant gene expression regulation. Discriminating the modified expression patterns driving tumor biology from the many that have no or little contribution is important for understanding cancer molecular basis. Recurrent deregulation patterns observed in multiple cancer types are enriched for such driver events. Here, we studied splicing alterations in hundreds of matched tumor and normal RNA-seq samples of eight solid cancer types. We found hundreds of cassette exons for which splicing was altered in multiple cancer types and identified a set of highly frequent altered splicing events. Specific splicing regulators, including RBFOX2, MBNL1/2 and QKI, appear to account for many splicing alteration events in multiple cancer types. Together, our results provide a first global analysis of regulated splicing alterations in cancer and identify common events with a potential causative role in solid tumor development.     

The continuously fed batch reactor for measuring microbial growth rates

The contiuously fed batch reactor (CFBR) is proposed as an alternative technique to the traditional chemostat and batch cultures, for measuring microbial growth rates. After reviewing the pitfalls which plague the conventional growth measurement techniques, the methodology for operating the CFBR to generate specific growth-rate-versus-substrate-concentration data is detailed. This information is extracted from the transient state of the CFBR where both the biomass and substrate concentration show extrema in time. It is suggested that the CFBR can be used for measuring microbial growth rates at low rates at low substrate concentrations where the chemostat method normally encounters difficulties.     

A quantitative stopped-flow fluorescence assay for measuring polymerase elongation rates

The measurement of nucleic acid polymerase elongation rates is often done via a lengthy experimental process involving radiolabeled substrates, quenched elongation experiments, electrophoretic product separation, and band quantitation. In this work, we describe an alternative real-time stopped-flow assay for obtaining kinetic parameters for elongation of extended sequences. The assay builds on our earlier PETE (polymerase elongation template element) assay designed for high-throughput screening purposes [S.P. Mestas, A.J. Sholders, O.B. Peersen, A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity, Anal. Biochem. 365 (2007) 194-200] and relies on measuring how long it takes a polymerase to reach the end of a defined length template. Using poliovirus polymerase and self-priming hairpin RNA substrates with 6- to 26-nt-long templating regions, we demonstrate that the assay can be used to determine V_max rates for elongation and apparent K_m values for nucleotide triphosphate (NTP) use. Modeling the reaction kinetics as a series of irreversible steps allows us to numerically fit the entire time-based dataset by properly accounting for the temporal distribution of intermediate species. This enables us to determine average elongation rates over heterogeneous templating regions that mimic viral genome substrates. The assay is easily extendable to other RNA and DNA polymerases, can accommodate secondary structures in the template, and can in principle be used for any enzyme traversing along an extended substrate.     

Image processing technique for measuring specific growth rates of Gibberella fujikuroi

Summary Two methods were compared for estimating of Gibberella fujikuroi grown with different proportions of glucose and starch. They were, =Ln2 (V_r/Le) on Petri dish (V_r= rate of tip extension and L_e= mean hyphal length) and, ₂=d (LnX)/dt in stirred fermenter. Values of ₁ and ₂ were in close agreement with each other.     

Evaluation of a fluorescent microparticle technique for measuring filtering rates of Daphnia

The paper demonstrates the applicability of fluorescent microparticles (FMP) for the determination of cladoceran feeding rates. For this purpose we examined the relation between filtering rate, ingestion rate, gut passage time and body length of Daphnia pulex at different food concentrations in laboratory experiments, and diurnal changes of filtering rates of Daphnia longispina in in situ experiments, using FMPs as tracer particles. Good agreement of our results with published data based on cell counts and radiotracer methods proves the suitability of the FMP-method. Potentials and limitations of the method are discussed.     

Tests for multivariate recurrent events in the presence of a terminal event

In studies involving diseases associated with high rates of mortality, trials are frequently conducted to evaluate the effects of therapeutic interventions on recurrent event processes terminated by death. In this setting, cumulative mean functions form a natural basis for inference for questions of a health economic nature, and Ghosh and Lin (2000) recently proposed a relevant class of test statistics. Trials of patients with cancer metastatic to bone, however, involve multiple types of skeletal complications, each of which may be repeatedly experienced by patients over their lifetime. Traditionally the distinction between the various types of events is ignored and univariate analyses are conducted based on a composite recurrent event. However, when the events have different impacts on patients' quality of life, or when they incur different costs, it can be important to gain insight into the relative frequency of the specific types of events and treatment effects thereon. This may be achieved by conducting separate marginal analyses with each analysis focusing on one type of recurrent event. Global inferences regarding treatment benefit can then be achieved by carrying out multiplicity adjusted marginal tests, more formal multiple testing procedures, or by constructing global test statistics. We describe methods for testing for differences in mean functions between treatment groups which accommodate the fact that each particular event process is ultimately terminated by death. The methods are illustrated by application to a motivating study designed to examine the effect of bisphosphonate therapy on the incidence of skeletal complications among patients with breast cancer metastatic to bone. We find that there is a consistent trend towards a reduction in the cumulative mean for all four types of skeletal complications with bisphosphonate therapy; there is a significant reduction in the need for radiation therapy for the treatment of bone. The global test suggests that bisphosphonate therapy significantly reduces the overall number of skeletal complications.     

The use of a rapid radiolabeling method for measuring ingestion rates of detritivores

Utilization of the organic components of senescent Spartina alterniflora Loisel detritus by the grass shrimp, Palaemonetes pugio Holthuis, was investigated in laboratory feeding studies. A ¹⁴C-dimethyl sulfate labeling procedure was utilized to label the sterilized detritus. The formed covalent bond is stable to autoclaving, freezing and extremes of pH. An ingestion rate of 2.0 × 10^?4CS·h^?1 was determined for P. pugio. Estimates for rate of incorporation, gross growth efficiency, and total biomass incorporated are presented. An inverse relationship was demonstrated between shrimp size and rates of ingestion and incorporation.     

A high-throughput method for measuring growth and loss rates in microalgal cultures

A miniaturized and low-cost assay for algal growth and loss rates, and estimation of compensation light was developed and optimized. Microalgal cultures were grown in white 96-well microplates to estimate specific growth rates at six temperatures, five salinities and eight light levels. Data from black 24-well microplates at six temperatures, five salinities and five light conditions were used in addition to estimate loss rates and compensation light. Absorption and reflection of light were different in the white and black microplates. Growth rates were estimated from daily in vivo fluorescence (IVF) measurements using a microplate reader fitted with a fluorometer. To validate the microplate algal growth assay, IVF was compared with cell counting by flow cytometry. Maximal growth rate for the test alga Pseudochattonella farcimen (Heterokonta) was estimated to 0.52?±?0.05 day^?1 at optimal temperatures ranging from 9 to 14°C and salinities 18–26 psu. Lowest value of compensation light as photosynthetic photon flux density (PPFD) was 4.2?±?1.2 μmol photons m^?2 s^?1, and lowest saturation light, 34.1?±?3.7 μmol photons m^?2 s^?1, was observed in the temperature range 5–11°C and salinity range 23–28 psu. Minimum loss rate was obtained at temperatures 5–8°C and salinities 26–31 psu. Blooms of P. farcimen have been recorded in nature under conditions similar to those minimizing loss rates rather than maximizing growth rates in this study. The microalgal assay described here allows for a large number of conditions to be tested, and accurate optimal conditions for growth and loss rates to be obtained.     

Suitability of 2-deoxyglucose for measuring initial rates of glucose uptake in isolated adipocytes

The suitability of [3H]-2-deoxyglucose from measuring initial rates of glucose uptake in isolated rat adipocytes was assessed using three approaches. Basal and insulin-stimulated rates of glucose uptake were directly compared in 2 sec and 5 min assays using [14C]-3-O-methylglucose, [3H]-2-deoxyglucose, and [3H]-D-glucose. Equilibrium kinetics of 2-deoxyglucose uptake were compared with those of 3-O-methylglucose through impairment of hexokinase activity by depleting cellular energy with 2,4-dinitrophenol. The equivalence of these glucose analogues in a dynamic system was assessed by measuring the lag time preceding insulin stimulation of glucose uptake, insulin activation rates, and the T 1/2 of insulin activation. Our results demonstrate that no fundamental difference exists in the initial transport of 3-O-methylglucose, 2-deoxyglucose, and D-glucose.     

The use of the Coulter Counter for measuring the feeding rates of daphnia magna

The feeding rate of Daphnia magna was determined by measuring the decrease of the Chlorella vulgaris concentration as animals were fed in a certain volume of algal suspension. The algal concentration was measured with a Coulter Counter. The results are quite comparable with the results of other methods reported in the literature. The advantage of this method is the possibility of studying the feeding rate over longer periods. The effect of the temperature on the filtering and feeding rate was determined. The highest values of both rates were found above 22°C.     

Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events

Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.