The likelihood ratio test for the two-component normal mixture problem: power and sample size analysis

We find, through simulation and modeling, an approximation to the alternative distribution of the likelihood ratio test for two-component mixtures in which the components have different means but equal variances. We consider the range of mixing proportions from 0.5 through .95. Our simulation results indicate a dependence of power on the mixing proportion when pi less than .2 and pi greater than .80. Our model results indicated that the alternative distribution is approximately noncentral chi-square, possibly with 2 degrees of freedom. Using this model, we estimate a sample of 40 is needed to have 50% power to detect a difference between means equal to 3.0 for mixing proportions between .2 and .8. The sample size increases to 50 when the mixing proportion is .90 (or .1) and 82 when the mixing proportion is .95 (or .05). This paper contains a complete table of sample sizes needed for 50%, 80%, and 90% power.     

Pattern of DNA synthesis and its effect on the classification of cells by flow cytometry

A flow cytometer produces a DNA distribution which contains information about the proportions of cells in various phases of the cell cycle. In this report we show that the form assumed for the rate of DNA synthesis in a cell plays an important part in the estimation of those proportions. We compare three forms for the rate of DNA synthesis, one of which is derived from knowledge of the mechanism of DNA replication, and find that they give substantially different estimates of the proportions of cells in the G1 and G2 + M phases.     

Multivariate bernoulli mixture models with application to postmortem tissue studies in schizophrenia

A novel mixture model is presented for repeated measurements in which correlation among repeated observations on the same subject is induced via correlated unobservable component indicators. The mixture components in our model are linear regressions, and the mixing proportions are logits with random effects. Inference is facilitated by sampling from the posterior distribution of the parameters via Markov chain Monte Carlo methods. The model is applied to a neuronal postmortem brain tissue study to examine the differences in neuron volumes between schizophrenic and control subjects.     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro

Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [3H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G0/G1, S, and G2/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [3H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [3H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G2 tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

A population balance model describing the cell cycle dynamics of myeloma cell cultivation

A multi-staged population balance model is proposed to describe the cell cycle dynamics of myeloma cell cultivation. In this model, the cell cycle is divided into three stages, i.e., G1, S, and G2M phases. Both DNA content and cell volume are used to differentiate each cell from other cells of the population. The probabilities of transition from G1 to S and division of G2M are assumed to be dependent on cell volume, and transition probability from S to G2M is determined by DNA content. The model can be used to simulate the dynamics of DNA content and cell volume distributions, phase fractions, and substrate and byproduct concentrations, as well as cell densities. Measurements from myeloma cell cultivations, especially the FACS data with respect to DNA distribution and cell fractions in different stages, are employed for model validation.     

Microfluidic components and bio-reactors for miniaturized bio-chip applications

In this paper miniaturized disposable micro/nanofluidic components applicable to bio chip, chemical analyzer and biomedical monitoring system, such as blood analysis, micro dosing system and cell experiment, etc are reported. This system includes various microfluidic components including a micropump, micromixer, DNA purification chip and single-cell assay chip. For low voltage and low power operation, a surface tension-driven micropump is presented, as well as a micromixer, which was implemented using MEMS technology, for efficient liquid mixing is also introduced. As bio-reactors, DNA purification and single-cell assay devices, for the extraction of pure DNA from liquid mixture or blood and for cellular engineering or high-throughput screening, respectively, are presented.     

Nucleus DNA content measurement methods features

We suggest a new theoretical method of the flow cytometry DNA histograms and apply it for Drosophila melanogaster imaginal discs cells. The model gives a possibility to determine the proportions of cells in G1, G2 (M) and S cell cycle phases. We show that the precision of G1 and G2 (M) DNA content measurements is limited by the precision of device zero signal arrangement. The usage of calculated device zero and dividing cells as the DNA content standards may improve the precision of DNA content measurements. We also compared the precisions of different DNA content methods and draw the conclusion that the current precisions of different methods are similar and lie within 2-6% interval.     

A computer model of spermatogenesis in the rat; correlation with flow cytometric data based on autoradiographic cell-cycle properties

A computer model of rat spermatogenesis was created, based on autoradiographic studies of durations of the phases of the cell cycle (G1, S, G2 and mitotic phases) of each germ-cell type. With this model it is possible to predict and to gain insight into the changes of the DNA content occurring during the normal process of spermatogenesis. The relative proportions of haploid, diploid, S phase and tetraploid germ cells with increasing age of the rats were calculated. Calculated and actual experimental flow cytometry data were compared to test the accuracy of the model, and these show good agreement. The present work demonstrates that single-parameter DNA analysis of testicular cells is primarily a reflection of germ cells in the spermatocyte and spermatid stages of development, and of non-germ cells. The FCM single-parameter DNA analysis of testicular cells is relatively insensitive to changes in the stem cell and spermatogonial stages of germ-cell development.     

Fluorometric determination of DNA and RNA in Chlamydomonas using ethidium bromide

By using the fluorescence enhancement of ethidium bromide bound to nuclei acid, a very rapid, simple and sensitive assay of DNA in the green alga Chlamydomonas has been devised. Total fluorescence (DNA + RNA) was determined by complex formation with ethidium bromide in a cell lysate made by mixing cell samples with lauroyl sarcosinate, EDTA and NaOH and incubating the mixture for 5 min at room temperature followed by neutralization. For determination of DNA the RNA was digested by incubating the cell sample in te alkaline lysis solution for 45 min at 60 degrees C followed by neutralization, and complex formation with ethidium bromide. Quenching of the fluorescence due to cellular pigments was corrected for using an internal DNA standard.     

Quantitation of total DNA per cell in an exponentially growing population using the diphenylamine reaction and flow cytometry

The diphenylamine assay used to estimate the absolute mass of DNA/cell as well as absolute differences in DNA content between cell populations is based upon the assumption that all of the cells are in the G0 or G1 phase of the DNA synthetic cycle. However, if cells are in exponential growth and synthesizing DNA, portions of the population will be in S or G2 phases and the diphenylamine assay will overestimate the total mass of DNA/cell. Conversely, flow cytometry (FCM) can estimate relative differences in total DNA/cell and the proportions of an exponentially growing population in G1, S, and G2 but cannot estimate absolute mass or differences in DNA/cell. In this report, we describe a methodology of combined diphenylamine and FCM assays of total DNA/cell which is applicable to any eukaryotic cell population. The method involves using the two assay methods concurrently and correcting the diphenylamine data for the FCM-derived distribution of the cells within the DNA synthetic cycle. The methodology was tested on single-cell-derived stocks of the obligate intracellular protozoan parasite Trypanosoma cruzi which displays marked but stable intraspecific heterogeneity.     

Flow-cytometric analysis of mixed cell populations using intermediate filament antibodies

Using two human tumour cell lines, T24 bladder carcinoma and Molt-4 leukemia, flow-cytometric DNA analysis of pure and mixed cell populations was performed using cellular cytokeratin content to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratin. Using cytokeratin content to gate DNA analysis, the same specificity and sensitivity of cellular DNA content and distribution measurement could be achieved by single-pass FCM analysis of a mixture of the two cell types as was seen when analysing pure populations of the two cell lines. This technique has broad applicability to FCM analysis of mixed populations composed of cells from different tissues of origin.     

Fitting mixture distributions to phenylthiocarbamide (PTC) sensitivity.

A technique for fitting mixture distributions to phenylthiocarbamide (PTC) sensitivity is described. Under the assumptions of Hardy-Weinberg equilibrium, a mixture of three normal components is postulated for the observed distribution, with the mixing parameters corresponding to the proportions of the three genotypes associated with two alleles A and a acting at a single locus. The corresponding genotypes AA, Aa, and aa are then considered to have separate means and variances. This paper is concerned with estimating the parameters of the model, and their standard errors, by using an application of the EM algorithm. This technique also caters for the fact that the sensitivity measurements are only known to lie between the endpoints of certain intervals and that the exact measurement of the attribute is not possible.     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication mitiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum

In Dictyostelium discoideum, the initial differentiation of cells is regulated by the phase of the cell cycle at starvation. Cells in S and early G2 (or with a low DNA content) have relatively high levels of cellular Ca2+ and display a prestalk tendency after starvation, whereas cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. We found that there is a correlation between cytosolic Ca2+ and cell cycle phase, with high Ca2+ levels being restricted to cells in the S and early G2 phases. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, it has been reported that in the rtoA mutant, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation. In contrast to the wild type, a disproportionately large fraction of rtoA amoebae fall into the high Ca2+ class, possibly due to an altered ability of this mutant to transport Ca2+.     

Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes

Rapidly proliferating, polyclonally stimulated mouse spleen lymphocytes were separated by density-gradient unit-gravity sedimentation. The following measurements were made on each fraction: the average intracellular water volume, the distribution of DNA content by flow microfluorometry, the rate of ³H-thymidine incorporation, and the intracellular pH. Fractions of cells with a small average intracellular volume were predominately in G0 or G1 phase of the cell cycle, while fractions of larger cells had higher proportions of cells in S or G2. Multiple regression analysis of the data for both T and B lymphocytes indicated that the intracellular pH of cells in G0, G1, or G2 is around pH 7.2, and that the intracellular pH of cells in S phase of the cell cycle is around pH 7.4.     

Analysis of DNA content in multidrug-resistant cells by image and flow cytometry

Abstract. Nuclear DNA content was assessed in multidrug-resistant (MDR) cells by image and flow cytometry. Two human MDR cell lines (K562-Dox and CEM-VLB) obtained by in vitro drug selection and overexpressing mdr1 gene were compared to their respective sensitive counterparts (K562 and CCRF-CEM) and to the MDR hamster LR73-R cell line obtained by transfection of mouse mdr1 cDNA. Both cell lines obtained by selection displayed a decreased DNA content, as measured by image cytometry after Feulgen staining, or by flow cytometry after staining with propidium iodide, ethidium bromide, or Hoechst 33342. This decrease was not accompanied by changes in cell cycle phase distribution of cells. Moreover, image cytometry of cells stained after various hydrolysis times in 5 M HCl indicated that MDR cells displayed the same hydrolysis kinetics and sensitivity as drug-sensitive cells with a well-preserved stoichiometry of the Feulgen reaction. LR73-R cells transfected with mdr1 cDNA exhibited only a very limited change in propidium iodide staining as compared with sensitive LR73 cells, suggesting that mdr1 gene overexpression alone could not account for the alterations in DNA content observed in the selected MDR cells.     

Exploiting the functional and taxonomic structure of genomic data by probabilistic topic modeling

In this paper, we present a method that enable both homology-based approach and composition-based approach to further study the functional core (i.e., microbial core and gene core, correspondingly). In the proposed method, the identification of major functionality groups is achieved by generative topic modeling, which is able to extract useful information from unlabeled data. We first show that generative topic model can be used to model the taxon abundance information obtained by homology-based approach and study the microbial core. The model considers each sample as a “document,” which has a mixture of functional groups, while each functional group (also known as a “latent topic”) is a weight mixture of species. Therefore, estimating the generative topic model for taxon abundance data will uncover the distribution over latent functions (latent topic) in each sample. Second, we show that, generative topic model can also be used to study the genome-level composition of “N-mer” features (DNA subreads obtained by composition-based approaches). The model consider each genome as a mixture of latten genetic patterns (latent topics), while each functional pattern is a weighted mixture of the “N-mer” features, thus the existence of core genomes can be indicated by a set of common N-mer features. After studying the mutual information between latent topics and gene regions, we provide an explanation of the functional roles of uncovered latten genetic patterns. The experimental results demonstrate the effectiveness of proposed method.     

Changes in the DNA content of amoebae of Dictyostelium discoideum during growth and development.

A fluorimetric assay has been used to determine the DNA content of amoebae of Dictyostelium discoideum during growth and development. Amoebae grown in axenic culture tended to be multinucleate and had a greater DNA content than amoebae grown with a bacterial substrate, which were mononucleate. During the first 10 h of development there was little change in the DNA content of amoebae grown with a bacterial substrate, but the average DNA content per cell in amoebae grown axenically decreased as the amoebae became virtually mononucleate. Amoebae at 10 h development that had been harvested during exponential axenic growth were divided into two populations by countercurrent distribution in a polymer two-phase system. DNA content indicated that one population was largely in the G2-phase of the cell cycle, whereas the other population was largely in the G1-phase. Similar results were obtained at 10 h development with amoebae harvested during the stationary phase of axenic growth, although these amoebae start development all in the G2-phase of the cell cycle. Spores had a low DNA content, indicating that they were in G1-phase. It is proposed that all amoebae in G2-phase after early development differentiate, after mitosis, into spores and that stalk cells are formed from amoebae that remain in G1-phase after 10 h development.     

A Stereological Approach for Estimation of Cellular Immunogold Labeling and Its Spatial Distribution in Oriented Sections Using the Rotator

Particulate gold labeling applied to ultrathin sections is a powerful approach for locating cellular proteins and lipids on thin sections of cellular structures and compartments. Effective quantitative methods now allow estimation of both density and distribution of gold labeling across aggregate organelles or compartment profiles. However, current methods generally use random sections of cells and tissues, and these do not readily present the information needed for spatial mapping of cellular quantities of gold label. Yet spatial mapping of gold particle labeling becomes important when cells are polarized or show internal organization or spatial shifts in protein/lipid localization. Here we have applied a stereological approach called the rotator to estimate cellular gold label and proportions of labeling over cellular compartments at specific locations related to a chosen cell axis or chosen cellular structures. This method could be used in cell biology for mapping cell components in studies of protein translocation, cell polarity, cell cycle stages, or component cell types in tissues. (J Histochem Cytochem 57:709–719, 2009)