Addressing inter-gene heterogeneity in maximum likelihood phylogenomic analysis: yeasts revisited

Phylogenomic approaches to the resolution of inter-species relationships have become well established in recent years. Often these involve concatenation of many orthologous genes found in the respective genomes followed by analysis using standard phylogenetic models. Genome-scale data promise increased resolution by minimising sampling error, yet are associated with well-known but often inappropriately addressed caveats arising through data heterogeneity and model violation. These can lead to the reconstruction of highly-supported but incorrect topologies. With the aim of obtaining a species tree for 18 species within the ascomycetous yeasts, we have investigated the use of appropriate evolutionary models to address inter-gene heterogeneities and the scalability and validity of supermatrix analysis as the phylogenetic problem becomes more difficult and the number of genes analysed approaches truly phylogenomic dimensions. We have extended a widely-known early phylogenomic study of yeasts by adding additional species to increase diversity and augmenting the number of genes under analysis. We have investigated sophisticated maximum likelihood analyses, considering not only a concatenated version of the data but also partitioned models where each gene constitutes a partition and parameters are free to vary between the different partitions (thereby accounting for variation in the evolutionary processes at different loci). We find considerable increases in likelihood using these complex models, arguing for the need for appropriate models when analyzing phylogenomic data. Using these methods, we were able to reconstruct a well-supported tree for 18 ascomycetous yeasts spanning about 250 million years of evolution.     

Mitochondrial heterogeneity during staurosporine-induced apoptosis in HL60 cells: analysis at the single cell and single organelle level

BACKGROUND: Apoptosis is a complex phenomenon during which several events occur. A growing interest exists on the role and functionality of mitochondria during this type of cell death. The responsibility of modifications in mitochondrial membrane potential (Delta Psi) in triggering apoptosis is under investigation. METHODS: We evaluated Delta Psi changes in HL60 cells treated with staurosporine (STS). Flow cytometry and confocal microscopy have been used to analyze samples stained with two Delta Psi-sensitive probes, JC-1 and MitoTrackertrade mark Red CMXRos. RESULTS: At the cellular level, we found heterogeneic behavior. Indeed, after STS treatment, some cells displayed typical markers of apoptosis and a collapse in Delta Psi. Others were apoptotic with no changes in Delta Psi, others changed Delta Psi without being apoptotic, and others were healthy. The same heterogeneic response to STS was found at the single organelle level. In a given cell, some mitochondria were depolarized whereas others were not. CONCLUSION: In this model of apoptosis, changes in Delta Psi can be different among cells of the same type and among different organelles of the same cell. The collapse in Delta Psi is thus a heterogeneic phenomenon that seems to be an ancillary event following the irreversible phase of the apoptotic process.     

GoIFISH: a system for the quantification of single cell heterogeneity from IFISH images

Molecular analysis has revealed extensive intra-tumor heterogeneity in human cancer samples, but cannot identify cell-to-cell variations within the tissue microenvironment. In contrast, in situ analysis can identify genetic aberrations in phenotypically defined cell subpopulations while preserving tissue-context specificity. GoIFISH is a widely applicable, user-friendly system tailored for the objective and semi-automated visualization, detection and quantification of genomic alterations and protein expression obtained from fluorescence in situ analysis. In a sample set of HER2-positive breast cancers GoIFISH is highly robust in visual analysis and its accuracy compares favorably to other leading image analysis methods. GoIFISH is freely available at www.sourceforge.net/projects/goifish/.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0442-y) contains supplementary material, which is available to authorized users.     

Modelling the individual cell lag phase. Isolating single cells: protocol development

AIMS: To develop a protocol to isolate single cells in wells of a microtitre plate, having a high certainty of individual cells, combined with a sufficient yield. METHODS AND RESULTS: Single cells were obtained using 1/2 dilution series in microtitre plates. Seventy-two Lactococcus lactis dilution series were checked by plate counting. When the last five columns of the plates were observed, the chance of having one single cell was 80%, while the yield was 75 wells containing cells. A simulation model confirmed these results. This method was compared with the commonly applied method. CONCLUSIONS: This method makes it possible to combine a higher chance of having one cell in a microtitre well with a slightly higher yield. SIGNIFICANCE AND IMPACT OF THE STUDY: A tool is developed to isolate single cells to provide a suitable base for investigating and modelling the individual cell lag phase.     

Limiting dilution analysis of T helper cell heterogeneity: a single class of T cell makes both IL 2 and IL 3

We have previously described a limiting dilution assay (LDA) for estimating the frequency, in the spleens of unprimed mice, of alloantigen-responsive cells that, together with their immediate clonal progeny, can produce interleukin 2 (IL 2) in short-term culture. In this paper, we provide further evidence that the limiting cell in these cultures is, in fact, the immediate precursor of the IL 2-producing cell rather than merely a participant in a multicellular cascade that ultimately leads to production of this lymphokine. We also demonstrate the usefulness of the LDA method for estimating the frequency of the limiting cell for production of IL 3. By analyzing the supernatants of these short-term microcultures for both IL 2 and IL 3, we show that essentially all wells that produce IL 3 also produce IL 2. Furthermore, the amount of IL 2 produced in any individual well correlates strongly with the IL 3 production in the same well. Our data suggest that both these lymphokines are produced by a single class of T cell, and further that the regulatory events that control IL 2 generation in allostimulated helper cells act to control IL 3 levels in parallel.     

Genome-wide linkage analysis of blood pressure under locus heterogeneity

We describe a method for mapping quantitative trait loci that allows for locus heterogeneity. A genome-wide linkage analysis of blood pressure was performed using sib-pair data from the Framingham Heart Study. Evidence of linkage was found on four markers (GATA89G08, GATA23D06, GATA14E09, and 049xd2) at a significance level of 0.01. Two of them (GATA14E09 and 049xd2) seem to overlap with linkage signals reported previously, while the other two are not linked to any known signals.     

New approaches for the analysis of mast cell maturation, heterogeneity, and function

Mast cells are regarded as potentially critical participants in a wide variety of important biological processes. Yet it has been difficult to ascertain the precise contribution of mast cells to many of these reactions. In part, this reflects recognition that different populations of mast cells may vary in phenotype and therefore may express different functions. Another problem has been the lack of suitable model systems for identifying and quantitating the mast cell's unique roles in biological processes in intact animals. This review will outline the development of promising new approaches for analyzing mouse mast cell differentiation and phenotypic heterogeneity and for studying the roles of mast cells in vivo.     

Drug delivery systems for oestrogenic hormones and antagonists: the need for selective targeting in estradiol-dependent cancers

The pleiotropic activity of oestrogens and their mechanism of action via their binding to the two oestrogen receptors alpha (ER alpha) and beta (ER beta) subtypes in the different tissues where oestrogens exert their action have been briefly described. The fate of these compounds trapped into different galenic forms is discussed with regard to their therapeutic applications. Firstly, the advantages and disadvantages of the different forms (pills, i.v. forms and transdermal patches) used in contraception are compared. Secondly, the therapeutic use of formulated oestrogens for the post-menopausal hormone replacement therapy (HRT) is analysed through the various results obtained in different trials. The link between HRT and the risks of breast cancer and cardiovascular disease is underlined. Finally, comparing the activity of selective oestrogen receptor modulators such as tamoxifen and pure anti-oestrogens such as RU58668 and ICI182780, we analysed the reasons leading to the need for a tumor targeting of the latters, but not of the former for the treatment of oestrogen-dependent breast cancer. Different injectable and biodegradable formulations, that lead to a remarkable anti-tumor efficiency in xenografts, have been recently developed and we believe that they may represent promising new administration ways of added therapeutic values for anti-oestrogens. Such devices could be extended to the delivery of other anti-cancer drugs with more aggressive activities than anti-oestrogens.     

Methods for the study of liver cell heterogeneity

A large number of histological, histochemical and biochemical techniques are available for studying liver cell heterogeneity. Structural differences are recognized by morphometric analyses of electron micrographs. The zonal heterogeneity of enzyme activities can be demonstrated by histochemistry and more precisely by ultramicrobiochemical assays in microdissected periportal and perivenous tissue. Immunohistochemistry is useful for quantifying and localizing proteins, especially isoenzymes, without depending on their biological activity. The zonal quantification of specific mRNA can be achieved by in situ hybridization. The different structural and enzymic equipment of periportal and perivenous tissue found by these techniques has led to the concept of metabolic zonation. This hypothesis can be confirmed by determination of metabolic rates in perfused liver after selective zonal damage, in separated periportal and perivenous hepatocytes as well as in periportal and perivenous tissue of perfused liver by non-invasive techniques.     

Density-dependence by habitat heterogeneity: individual quality versus territory quality

The aim of this comment is to review the ecological issues concerning the role of individual and habitat heterogeneity as possible mechanisms explaining density-dependent fecundity in animal populations. Our intention is to discuss different approaches to determine whether or not studied populations are subjected to density-dependent processes and which mechanisms are involved. We show that because individual quality (e.g. measured in terms of age of breeding individuals) and territory quality can be correlated, untangling both effects on fecundity is frequently a difficult task. We discuss the misuse of statistical methods and other problems related to the specific characteristics of the studied populations that can have a strong influence on the conclusions reached by researchers working in this field.     

Addressing single molecules of a thin magnetic film

Electron spin noise-scanning tunneling microscopy (ESN-STM) represents the most promising technique for single spin centres addressing on surfaces. After a brief introduction on the results previously obtained by using this technique for the detection of both isolated molecules and small aggregates of BDPA, DPPH and TTM free radicals, we discuss here our improved results in the single-molecule addressing of thio-functionalized nitronyl nitroxide radicals (NNRs) chemically bound to gold. ESN-STM spectra of NNRs self-assembled monolayers (SAMs) on Au(1 1 1) surfaces are reported together with the crystal structure of the studied radical NN–Ph–CH₂SMe, considered up to now as the best aromatic NNR for the obtainment of thin magnetic films.     

Illuminating intracellular signaling and molecules for single cell analysis

Fluorescent and bioluminescent proteins are now widely used for detection of small molecules and various intracellular events ranging from protein conformational change to cell death in living cells. To analyze the dynamics of molecular processes in real time at the level of single cells, engineered protein-based probes with higher sensitivity and selectivity are required. The probes can be entirely genetically encoded and can comprise fusions of different proteins or domains. This review specifically examines basic concepts of designing genetically encoded fluorescent and bioluminescent probes developed in the past decade, highlighting some potential applications for basic research and for drug discovery.