A statistical pattern recognition approach for determining cellular viability and lineage phenotype in cultured cells and murine bone marrow.

BACKGROUND: Cellular binding of annexin V and membrane permeability to 7-aminoactinomycin D (7AAD) are important tools for studying apoptosis and cell death by flow cytometry. Combining viability markers with cell surface marker expression is routinely used to study various cell lineages. Current classification methods using strict thresholds, or "gates," on the fluorescent intensity of these markers are subjective in nature and may not fully describe the phenotypes of interest. We have developed objective criteria for phenotypic boundary recognition through the application of statistical pattern recognition. This task was achieved using artificial neural networks (ANNs) that were trained to recognize subsets of cells with known phenotypes, and then used to determine decision boundaries based on statistical measures of similarity. This approach was then used to test the hypothesis that erythropoietin (EPO) inhibits apoptosis and cell death in erythroid precursor cells in murine bone marrow. METHODS: Our method was developed for classification of viability using an in vitro cell system and then applied to an ex vivo analysis of murine late-stage erythroid progenitors. To induce apoptosis and cell death in vitro, an EPO-dependent human leukemic cell line, UT-7(EPO) cells were incubated without recombinant human erythropoietin (rhEPO) for 72 h. Five different ANNs were trained to recognize live, apoptotic, and dead cells using a "known" subset of the data for training, and a K-fold cross validation procedure for error estimation. The ANNs developed with the in vitro system were then applied to classify cells from an ex vivo study of rhEPO treated mice. Tg197 (human tumor necrosis-alpha transgenic mice, a model of anemia of chronic disease) received a single s.c. dose of 10,000 U/kg rhEPO and femoral bone marrow was collected 1, 2, 4, and 8 days after dosing. Femoral bone marrow cells were stained with TER-119 PE, CD71 APC enable identification of erythroid precursors, and annexin V FITC and 7AAD to identify the apoptotic and dead cells. During classification forward and side angle light scatter were also input to all pattern recognition systems. RESULTS: Similar decision boundaries between live, apoptotic, and dead cells were consistently identified by the neural networks. The best performing network was a radial basis function multi-perceptron that produced an estimated average error rate of 4.5% +/- 0.9%. Using these boundaries, the following results were reached: depriving UT-7(EPO) cells of rhEPO induced apoptosis and cell death while the addition of rhEPO rescued the cells in a dose-dependent manner. In vivo, treatment with rhEPO resulted in an increase of live erythroid cells in the bone marrow to 119.8% +/- 9.8% of control at the 8 day time point. However, a statistically significant transient increase in TER-119(+) CD71(+) 7AAD(+) dead erythroid precursors was observed at the 1 and 2 day time points with a corresponding decrease in TER-119(+) CD71(+) 7AAD(-) Annexin V(-) live erythroid precursors, and no change in the number of TER-119(+) CD71(+) annexin V(+) 7AAD(-) apoptotic erythroid precursors in the bone marrow. CONCLUSIONS: A statistical pattern recognition approach to viability classification provides an objective rationale for setting decision boundaries between "positive" and "negative" intensity measures in cytometric data. Using this approach we have confirmed that rhEPO inhibits apoptosis and cell death in an EPO dependent cell line in vitro, but failed to do so in vivo, suggesting EPO may not act as a simple antiapoptotic agent in the bone marrow. Rather, homeostatic mechanisms may regulate the pharmacodynamic response to rhEPO.     

A cross-cancer metastasis signature in the microRNA–mRNA axis of paired tissue samples

In the progression of cancer, cells acquire genetic mutations that cause uncontrolled growth. Over time, the primary tumour may undergo additional mutations that allow for the cancerous cells to spread throughout the body as metastases. Since metastatic development typically results in markedly worse patient outcomes, research into the identity and function of metastasis-associated biomarkers could eventually translate into clinical diagnostics or novel therapeutics. Although the general processes underpinning metastatic progression are understood, no clear cross-cancer biomarker profile has emerged. However, the literature suggests that some microRNAs (miRNAs) may play an important role in the metastatic progression of several cancer types. Using a subset of The Cancer Genome Atlas (TCGA) data, we performed an integrated analysis of mRNA and miRNA expression with paired metastatic and primary tumour samples to interrogate how the miRNA–mRNA regulatory axis influences metastatic progression. From this, we successfully built mRNA- and miRNA-specific classifiers that can discriminate pairs of metastatic and primary samples across 11 cancer types. In addition, we identified a number of miRNAs whose metastasis-associated dysregulation could predict mRNA metastasis-associated dysregulation. Among the most predictive miRNAs, we found several previously implicated in cancer progression, including miR-301b, miR-1296, and miR-423. Taken together, our results suggest that metastatic samples have a common cross-cancer signature when compared with their primary tumour pair, and that these miRNA biomarkers can be used to predict metastatic status as well as mRNA expression.

     

Chemical investigation of drug-like compounds from the Australian tree,Neolitsea dealbata

Two of the four parameters in the ‘rule of five’, molecular weight and log P, which can be detected and predicted by mass spectrometry and compound retention on reversed-phase HPLC, were used as guidelines in natural product isolation. A new aporphine alkaloid, (6aR)-normecambroline (1), was isolated from the bark of Neolitsea dealbata (R. Br.) Merr. Its structure was determined on the basis of NMR, MS and CD analysis. It is the first time the absolute configuration of the roemerine-N-oxide was assigned for both roemerine-N_α-oxide (3) and roemerine-N_β-oxide (4). Physico-chemical property evaluation demonstrated all alkaloids had no Lipinski violation. Compound 1 inhibited selectively against cervical cancer cells (HeLa) with an IC₅₀ of 4.0 μM.     

Characterization of the Drosophila caspase, DAMM

Caspases are main effectors of apoptosis in metazoans. Genome analysis indicates that there are seven caspases in Drosophila, six of which have been previously characterized. Here we describe the cloning and characterization of the last Drosophila caspase, DAMM. Similar to mammalian effector caspases, DAMM lacks a long prodomain. We show that the DAMM precursor, along with the caspases DRONC and DECAY, is partially processed in cells undergoing apoptosis. Recombinant DAMM produced in Escherichia coli shows significant catalytic activity on a pentapeptide caspase substrate. Low levels of damm mRNA are ubiquitously expressed in Drosophila embryos during early stages of development. Relatively high levels of damm mRNA are detected in larval salivary glands and midgut, and in adult egg chambers. Ectopic expression of DAMM in cultured cells induces apoptosis, and similarly, transgenic overexpression of DAMM, but not of a catalytically inactive DAMM mutant, in Drosophila results in a rough eye phenotype. We demonstrate that expression of the catalytically inactive DAMM mutant protein significantly suppresses the rough eye phenotype due to the overexpression of HID, suggesting that DAMM may be required in a hid-mediated cell death pathway.     

A novel osteoblast-derived C-type lectin that inhibits osteoclast formation

We have cloned and expressed murine osteoclast inhibitory lectin (mOCIL), a 207-amino acid type II transmembrane C-type lectin. In osteoclast formation assays of primary murine calvarial osteoblasts with bone marrow cells, antisense oligonucleotides for mOCIL increased tartrate-resistant acid phosphatase-positive mononucleate cell formation by 3-5-fold, whereas control oligonucleotides had no effect. The extracellular domain of mOCIL, expressed as a recombinant protein in Escherichia coli, dose-dependently inhibited multinucleate osteoclast formation in murine osteoblast and spleen cell co-cultures as well as in spleen cell cultures treated with RANKL and macrophage colony-stimulating factor. Furthermore, mOCIL acted directly on macrophage/monocyte cells as evidenced by its inhibitory action on adherent spleen cell cultures, which were depleted of stromal and lymphocytic cells. mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation and resulted in 70% inhibition during the differentiation phase. Osteoblast OCIL mRNA expression was enhanced by parathyroid hormone, calcitriol, interleukin-1alpha and -11, and retinoic acid. In rodent tissues, Northern blotting, in situ hybridization, and immunohistochemistry demonstrated OCIL expression in osteoblasts and chondrocytes as well as in a variety of extraskeletal tissues. The overlapping tissue distribution of OCIL mRNA and protein with that of RANKL strongly suggests an interaction between these molecules in the skeleton and in extraskeletal tissues.     

Migratory costs and the evolution of egg size and number in introduced and indigenous salmon populations

The trade-off between reproductive investment and migration should be an important factor shaping the evolution of life-history traits among populations following their radiation into habitats with different migratory costs and benefits. An experimentally induced difference in migratory rigor for families of chinook salmon (Oncorhynchus tshawytscha), of approximately 86 km and 413 m elevation, exacted a cost to somatic energy reserves (approximately 17% reduction in metabolizable mass) and ovarian investment (13.7% reduction in ovarian mass). This cost was associated with a reduction in egg size and paralleled the phenotypic pattern of divergence between two introduced New Zealand populations of common origin, presently breeding at sites with different migration distances. The genetic pattern of divergence of these same populations, detected under common rearing, was consistent with compensation for migratory costs (the population that migrates farther invested more in ovarian mass), but egg number more than egg size was associated with this evolution. These evolutionary patterns are consistent with what is known of the inheritance of these traits and with trade-offs and constraints favoring initial evolution in offspring number over offspring size. Analysis of egg number-size patterns of other Pacific salmon populations in their native range supported the hypothesis that migration strongly influences patterns of reproductive allocation, favoring a higher ratio of egg number to egg size with greater migration distance.     

POPULATION VARIABILITY IN DANTHONIA CAESPITOSA (GRAMINEAE) IN RESPONSES TO INCREASING DENSITY UNDER THREE TEMPERATURE REGIMES

The objective of this research was to compare the responses to increasing density of different natural populations of Danthonia caespitosa, a perennial grass of southern Australia, under different temperature regimes. Seeds were collected from four populations along a 167-km N–S transect beginning north of Deniliquin, N.S.W., and ending near Heathcote, Victoria. Seedlings from each population were planted in loamy sand at three densities and grown under day/night temperature regimes of 15/10, 24/19, and 33/28 C in the Canberra Ceres Phytotron. Results indicate that one natural population (or one temperature regime) can not be used to characterize this species' responses to increasing density. The effects of density and temperature on tillering, plant height, leaf length, and leaf width varied significantly among populations, so that populations achieved comparable shoot weights by different relative responses to density. Variability in biomass among individuals (as indicated by coefficients of variation) increased from low to intermediate density; however, size variability at the highest density was either greater, lesser, or approximately the same as that of plants at the intermediate density, depending upon the population and the temperature regime. The combinations of density and temperature which produced the maximum shoot growth per pot varied markedly among populations; at 24/19 C, populations from frequently disturbed areas of low perennial plant cover had greater shoot weights per pot at the intermediate density, while those populations from more stable grassland sites had their greatest shoot weights at the highest density. Flowering occurred in all populations at the low density in 15/10 C; however, only for the population from the ungrazed grassland was there any flowering in the other temperature regimes or at higher densities. It is concluded that a “response to density stress” could be differentially described for each population at each temperature regime, even though the four study populations represented only 1/10 of the latitudinal range of D. caespitosa.     

The ends of a continuum: genetic and temperature-dependent sex determination in reptiles

Two prevailing paradigms explain the diversity of sex-determining modes in reptiles. Many researchers, particularly those who study reptiles, consider genetic and environmental sex-determining mechanisms to be fundamentally different, and that one can be demonstrated experimentally to the exclusion of the other. Other researchers, principally those who take a broader taxonomic perspective, argue that no clear boundaries exist between them. Indeed, we argue that genetic and environmental sex determination in reptiles should be seen as a continuum of states represented by species whose sex is determined primarily by genotype, species where genetic and environmental mechanisms coexist and interact in lesser or greater measure to bring about sex phenotypes, and species where sex is determined primarily by environment. To do otherwise limits the scope of investigations into the transition between the two and reduces opportunities to use studies of reptiles to advance understanding of vertebrate sex determination generally.