Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.     

Detecting and Measuring Selection from Gene Frequency Data

The recent advent of high-throughput sequencing and genotyping technologies makes it possible to produce, easily and cost effectively, large amounts of detailed data on the genotype composition of populations. Detecting locus-specific effects may help identify those genes that have been, or are currently, targeted by natural selection. How best to identify these selected regions, loci, or single nucleotides remains a challenging issue. Here, we introduce a new model-based method, called SelEstim, to distinguish putative selected polymorphisms from the background of neutral (or nearly neutral) ones and to estimate the intensity of selection at the former. The underlying population genetic model is a diffusion approximation for the distribution of allele frequency in a population subdivided into a number of demes that exchange migrants. We use a Markov chain Monte Carlo algorithm for sampling from the joint posterior distribution of the model parameters, in a hierarchical Bayesian framework. We present evidence from stochastic simulations, which demonstrates the good power of SelEstim to identify loci targeted by selection and to estimate the strength of selection acting on these loci, within each deme. We also reanalyze a subset of SNP data from the Stanford HGDP–CEPH Human Genome Diversity Cell Line Panel to illustrate the performance of SelEstim on real data. In agreement with previous studies, our analyses point to a very strong signal of positive selection upstream of the LCT gene, which encodes for the enzyme lactase–phlorizin hydrolase and is associated with adult-type hypolactasia. The geographical distribution of the strength of positive selection across the Old World matches the interpolated map of lactase persistence phenotype frequencies, with the strongest selection coefficients in Europe and in the Indus Valley.     

Continental scale analysis of bird migration timing: influences of climate and life history traits—a generalized mixture model clustering and discriminant approach

There is substantial evidence of climate-related shifts to the timing of avian migration. Although spring arrival has generally advanced, variable species responses and geographical biases in data collection make it difficult to generalise patterns. We advance previous studies by using novel multivariate statistical techniques to explore complex relationships between phenological trends, climate indices and species traits. Using 145 datasets for 52 bird species, we assess trends in first arrival date (FAD), last departure date (LDD) and timing of peak abundance at multiple Australian locations. Strong seasonal patterns were found, i.e. spring phenological events were more likely to significantly advance, while significant advances and delays occurred in other seasons. However, across all significant trends, the magnitude of delays exceeded that of advances, particularly for FAD (+22.3 and ?9.6 days/decade, respectively). Geographic variations were found, with greater advances in FAD and LDD, in south-eastern Australia than in the north and west. We identified four species clusters that differed with respect to species traits and climate drivers. Species within bird clusters responded in similar ways to local climate variables, particularly the number of raindays and rainfall. The strength of phenological trends was more strongly related to local climate variables than to broad-scale drivers (Southern Oscillation Index), highlighting the importance of precipitation as a driver of movement in Australian birds.     

Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

Ancient DNA (aDNA) research has long depended on the power of PCR to amplify trace amounts of surviving genetic material from preserved specimens. While PCR permits specific loci to be targeted and amplified, in many ways it can be intrinsically unsuited to damaged and degraded aDNA templates. PCR amplification of aDNA can produce highly-skewed distributions with significant contributions from miscoding lesion damage and non-authentic sequence artefacts. As traditional PCR-based approaches have been unable to fully resolve the molecular nature of aDNA damage over many years, we have developed a novel single primer extension (SPEX)-based approach to generate more accurate sequence information. SPEX targets selected template strands at defined loci and can generate a quantifiable redundancy of coverage; providing new insights into the molecular nature of aDNA damage and fragmentation. SPEX sequence data reveals inherent limitations in both traditional and metagenomic PCR-based approaches to aDNA, which can make current damage analyses and correct genotyping of ancient specimens problematic. In contrast to previous aDNA studies, SPEX provides strong quantitative evidence that C > U-type base modifications are the sole cause of authentic endogenous damage-derived miscoding lesions. This new approach could allow ancient specimens to be genotyped with unprecedented accuracy.     

Association of Mc1r variants with ecologically relevant phenotypes in the European ocellated lizard, Lacerta lepida

A comprehensive knowledge on the genetic basis of coloration is crucial to understand how new colour phenotypes arise and how they contribute to the emergence of new species. Variation in melanocortin‐1 receptor (Mc1r), a gene that has been reported as a target for repeated evolution in a wide range of vertebrate taxa, was assessed in European ocellated lizards (Lacerta lepida) to search for associations with melanin‐based colour phenotypes. Lacerta lepida subspecies’ distribution is associated with the three major bio‐climatic regions in the Iberian Peninsula. A nonconserved and derived substitution (T162I) was associated with the L. l. nevadensis phenotype (prevalence of brown scales). Another substitution (S172C) was associated with the presence of black scales in both L. l. lepida and L. l. iberica, but no mutations were found to be associated with the higher proportion of black in L. l. iberica. Extensive genotyping of Mc1r along the contact zone between L. l. nevadensis and L. l. lepida revealed low gene flow (only two hybrids detected). The implications of these findings are discussed in the context of previous knowledge about the evolutionary history of ocellated lizards.