Chris D. Lowe

DNA-Dependent Protein Kinase Is a Therapeutic Target and an Indicator of Poor Prognosis in B-Cell Chronic Lymphocytic Leukemia

Purpose: del(17p), del(11q), and associated p53 dysfunction predict for short survival and chemoresistance in B-cell chronic lymphocytic leukemia (CLL). DNA-dependent protein kinase (DNA-PK) is activated by DNA damage and mediates DNA double-strand break repair. We hypothesized that inhibiting DNA-PK would sensitize CLL cells to drug-induced DNA damage and that this approach could increase the therapeutic index of agents used to treat CLL. Experimental Design: Fifty-four CLL cases were characterized for poor prognosis markers [del(17p), del(11q), CD38, and ZAP-70]. In selected cases, DNA-PK catalytic subunit (DNA-PKcs) expression and activity and p53 function were also measured. Ex vivo viability assays established sensitivity to fludarabine and chlorambucil and also tested the ability of a novel DNA-PK inhibitor (NU7441) to sensitize CLL cells to these drugs. The effects of NU7441 on fludarabine-induced DNA damage repair were also assessed (Comet assays and detection of γH2AX). Results: DNA-PKcs levels correlated with DNA-PK activity and varied 50-fold between cases but were consistently higher in del(17p) (P = 0.01) and del(11q) cases. NU7441 sensitized CLL cells to chlorambucil and fludarabine, including cases with del(17p), del(11q), p53 dysfunction, or high levels of DNA-PKcs. NU7441 increased fludarabine-induced double-strand breaks and abrogated drug-induced autophosphorylation of DNA-PKcs at Ser2056. High DNA-PK levels predicted for reduced treatment-free interval. Conclusions: These data validate the concept of targeting DNA-PKcs in poor risk CLL, and demonstrate a mechanistic rationale for use of a DNA-PK inhibitor. The novel observation that DNA-PKcs is overexpressed in del(17p) and del(11q) cases indicates that DNA-PK may contribute to disease progression in CLL.

Patterns of Genetic Diversity in the Marine Heterotrophic Flagellate Oxyrrhis marina (Alveolata: Dinophyceae)

Oxyrrhis marina is an important model in ecological studies of free-living protists. Despite this, O. marina has rarely been studied in the environment and no explicit distributional studies exist. Further, phylogenetic data for a small number of isolates indicate that O. marina constitutes two divergent lineages. Here, we quantify phylogenetic variation between 58 globally distributed O. marina isolates using 5.8S--internal transcribed spacer 1 and 2 rDNA (5.8S ITS) and cytochrome c oxidase I (COI) partial sequences. 5.8S ITS and COI phylogenies both partitioned O. marina into four clades, which formed two lineages; mean sequence identity for 5.8S ITS and COI respectively was approximately 40 and 90% between these two lineages. Sequence identities for 5.8S ITS/ COI between clades within lineages were 66.3/99.4% (lineage 1: clade 1 vs 2) and 42.3/99.1% (lineage 2: clade 3 vs 4). rDNA mutation rates in O. marina appear to be abnormally high and were not interpreted in a species delineation context. Based on variation in COI sequence and comparisons with other protists, we suggest that O. marina lineages may constitute two species. In a geographic context, evidence of spatial restriction but also extensive overlap between O. marina clades occurred. Further, clade abundances varied considerably: clades 1 and 2 (belonging to one lineage) were abundant and widespread; in contrast, clades 3 and 4 (belonging to the second lineage) were rare and spatially restricted (occurring only in the Mediterranean or in culture collection). There is need for further phylogenetic and taxonomic studies to assess species delineation in O. marina, and for the application of high resolution genetic markers to resolve processes driving genetic diversity in this important model organism.

Field estimates of reproductive success in a model insect: behavioural surrogates are poor predictors of fitness

Understanding, and therefore measuring, factors that determine fitness is a central problem in evolutionary biology. We studied a natural population of Coenagrion puella (Odonata: Zygoptera) over two entire breeding seasons, with over a thousand individuals uniquely marked and genotyped, and all mating events at the rendezvous site recorded. Using a parentage analysis, fitness of individuals in the first generation was quantified as the numbers of offspring that survived to maturity. Although mating behaviour can be predicted by environmental and demographical variables, the numbers of mature offspring produced (fitness) cannot, and crucially, are poorly correlated with behavioural observations of mating. While fitness of both sexes was positively related to mating behaviour and to females ectoparasite burden, these behavioural observations explained little more variance in offspring production than environmental and demographical variables. Thus, we demonstrate that behavioural measures of reproductive success are not necessarily reliable estimates of fitness in natural populations.

The rise of model protozoa

It is timely to evaluate the role of protozoa as model organisms given their diversity, abundance and versatility as well as the economic and ethical pressures placed on animal-based experimentation. We first define the term model organism and then examine through examples why protozoa make good models. Our examples reflect major issues including evolution, ecology, population and community biology, disease, the role of organelles, ageing, space travel, toxicity and teaching. We conclude by recognising that although protozoa may in some cases not completely mimic tissue- or whole-animal-level processes, they are extremely flexible and their use should be embraced. Finally, we offer advice on obtaining emergent model protozoa.

High Genetic Diversity and Fine-Scale Spatial Structure in the Marine Flagellate Oxyrrhis marina (Dinophyceae) Uncovered by Microsatellite Loci

Free-living marine protists are often assumed to be broadly distributed and genetically homogeneous on large spatial scales. However, an increasing application of highly polymorphic genetic markers (e.g., microsatellites) has provided evidence for high genetic diversity and population structuring on small spatial scales in many free-living protists. Here we characterise a panel of new microsatellite markers for the common marine flagellate Oxyrrhis marina. Nine microsatellite loci were used to assess genotypic diversity at two spatial scales by genotyping 200 isolates of O. marina from 6 broad geographic regions around Great Britain and Ireland; in one region, a single 2 km shore line was sampled intensively to assess fine-scale genetic diversity. Microsatellite loci resolved between 1–6 and 7–23 distinct alleles per region in the least and most variable loci respectively, with corresponding variation in expected heterozygosities (He) of 0.00–0.30 and 0.81–0.93. Across the dataset, genotypic diversity was high with 183 genotypes detected from 200 isolates. Bayesian analysis of population structure supported two model populations. One population was distributed across all sampled regions; the other was confined to the intensively sampled shore, and thus two distinct populations co-occurred at this site. Whilst model-based analysis inferred a single UK-wide population, pairwise regional FST values indicated weak to moderate population sub-division (0.01–0.12), but no clear correlation between spatial and genetic distance was evident. Data presented in this study highlight extensive genetic diversity for O. marina; however, it remains a substantial challenge to uncover the mechanisms that drive genetic diversity in free-living microorganisms.

The transcriptome of the novel dinoflagellate Oxyrrhis marina (Alveolata: Dinophyceae): response to salinity examined by 454 sequencing

BackgroundThe heterotrophic dinoflagellate Oxyrrhis marina is increasingly studied in experimental, ecological and evolutionary contexts. Its basal phylogenetic position within the dinoflagellates make O. marina useful for understanding the origin of numerous unusual features of the dinoflagellate lineage; its broad distribution has lent O. marina to the study of protist biogeography; and nutritive flexibility and eurytopy have made it a common lab rat for the investigation of physiological responses of marine heterotrophic flagellates. Nevertheless, genome-scale resources for O. marina are scarce. Here we present a 454-based transcriptome survey for this organism. In addition, we assess sequence read abundance, as a proxy for gene expression, in response to salinity, an environmental factor potentially important in determining O. marina spatial distributions.ResultsSequencing generated ~57 Mbp of data which assembled into 7, 398 contigs. Approximately 24% of contigs were nominally identified by BLAST. A further clustering of contigs (at ≥ 90% identity) revealed 164 transcript variant clusters, the largest of which (Phosphoribosylaminoimidazole-succinocarboxamide synthase) was composed of 28 variants displaying predominately synonymous variation. In a genomic context, a sample of 5 different genes were demonstrated to occur as tandem repeats, separated by short (~200-340 bp) inter-genic regions. For HSP90 several intergenic variants were detected suggesting a potentially complex genomic arrangement. In response to salinity, analysis of 454 read abundance highlighted 9 and 20 genes over or under expressed at 50 PSU, respectively. However, 454 read abundance and subsequent qPCR validation did not correlate well - suggesting that measures of gene expression via ad hoc analysis of sequence read abundance require careful interpretation.ConclusionHere we indicate that tandem gene arrangements and the occurrence of multiple transcribed gene variants are common and indicate potentially complex genomic arrangements in O. marina. Comparison of the reported data set with existing O. marina and other dinoflagellates ESTs indicates little sequence overlap likely as a result of the relatively limited extent of genome scale sequence data currently available for the dinoflagellates. This is one of the first 454-based transcriptome surveys of an ancestral dinoflagellate taxon and will undoubtedly prove useful for future comparative studies aimed at reconstructing the origin of novel features of the dinoflagellates.

Redescription of Strombidium oculatum Gruber 1884 (Ciliophora, Oligotrichia)

Abstract The marine, tide pool-dwelling ciliate Stombidium oculatum was redescribed using live, stained, SEM, and TEM material prepared from samples collected from pools on the Isle of Man (Irish Sea) and Brittany (France). Also, we reviewed the older German and French works that reported on ciliates collected in the Mediterranean and Brittany, respectively. The Brittany and Isle of Man populations of the ciliate were considered identical. Some morphological and behavioural differences exist between the Brittany-Isle of Man populations and the Mediterranean populations, but they were insufficient to distinguish different taxa. Thus, taxa from all three locations were considered to be conspecific. Key features used to describe the ciliate were: morphology and ultrastructure of the free-swimming ciliate; cyst morphology; presence of mixotrophic-chloroplasts; presence of an eye spot composed of stigma obtained from chlorophyte prey; division, morphogenesis, and nuclear structure; live observations and behaviour, including the encystment-excystment cycle. Based on morphological and behavioural characteristics the taxon was distinguished from other similar species, and a neotype has been designated as no type material exists.

Empirical evidence of senescence in adult damselflies (Odonata: Zygoptera).

1. Age-dependent increases in mortality have been documented in a variety of species of insect under laboratory conditions. However, while strong statistical evidence has been presented for senescence in vertebrate populations in the wild, we know little about the rate and shape of senescence in wild populations of insects. 2. Odonates (damselflies and dragonflies) provide excellent candidate species for evaluating demographic senescence as they are large enough to be marked individually and they are easily re-sighted without recapture. The prevailing opinion - based entirely on qualitative examination of the declines in log numbers alive with time since marking - is that odonates exhibit age-independent daily survivorship. 3. Here, we examine mark-recapture data on the Azure Damselfly Coenagrion puella over two consecutive seasons. For the first time, we evaluate and compare the fit of quantitative models that not only account for weather-dependent daily variation in daily re-sighting rates, but also age-dependent variation in daily survivorship. 4. Models with age-dependent declines in daily survivorship provide a more parsimonious explanation for the data than similar models without these age-dependent effects. In general, models in which mortality increases in an exponential (Gompertz) fashion explain the mark-recapture sequences more efficiently than a range of alternative models, including those in which mortality increases as a power function (Weibull) or reaches a plateau (logistic). These results are indicative of a general senescent decline in physiological functioning, which is particularly marked after 15 days as a mature adult. 5. Weather (temperature, sun and precipitation) and initial mite load influenced the probability of daily re-sighting. Weather and mite load also influenced daily survivorship, but their effects differed between seasons. 6. Overall, fitting models with age as an explicit covariate demonstrates that odonates do indeed senesce. This contradicts previously held assumptions that Odonata do not exhibit age-dependent survivorship in the wild.

Strain-specific functional and numerical responses are required to evaluate impacts on predator-prey dynamics.

We use strains recently collected from the field to establish cultures; then, through laboratory studies we investigate how among strain variation in protozoan ingestion and growth rates influences population dynamics and intraspecific competition. We focused on the impact of changing temperature because of its well-established effects on protozoan rates and its ecological relevance, from daily fluctuations to climate change. We show, first, that there is considerable inter-strain variability in thermal sensitivity of maximum growth rate, revealing distinct differences among multiple strains of our model species Oxyrrhis marina. We then intensively examined two representative strains that exhibit distinctly different thermal responses and parameterised the influence of temperature on their functional and numerical responses. Finally, we assessed how these responses alter predator–prey population dynamics. We do this first considering a standard approach, which assumes that functional and numerical responses are directly coupled, and then compare these results with a novel framework that incorporates both functional and numerical responses in a fully parameterised model. We conclude that: (i) including functional diversity of protozoa at the sub-species level will alter model predictions and (ii) including directly measured, independent functional and numerical responses in a model can provide a more realistic account of predator–prey dynamics.

Reproductive timing and patterns of development for the damselfly Coenagrion puella in the field.

By a combination of detailed behavioral observations and molecular genetic approaches we have assessed development time, timing of first maturity, and the extent of genetic structure through the flying season in a wild population of the damselfly Coenagrion puella in England. This work provides the first estimate of development time (egg to mature adult) in the field based on individual damselflies. Development time was significantly longer for females than males. In contrast to reported laboratory studies, there was no difference in development times between different female color morphs. Development time ranged between 347 and 396 days and was negatively correlated with egg-laying date. As a result eggs laid early in one season reach adult maturity relatively late in the next; concurrently individuals developing from eggs laid late mature relatively early. We speculate that this pattern of development is a direct physiological response to seasonal environmental variation and results in reproductive synchrony within a population. Size, specifically hind wing length, declined with development time in males, but not in females. In one of the two years of the study there was evidence for weak clustering of related individuals during the reproductive season. This appeared to be the result of developmental synchronization within families: variance in timing of maturation was smaller in full-sib families than in half-sib families or randomly assigned unrelated groups.