Comparative transcriptomics of limb regeneration: Identification of conserved expression changes among three species of Ambystoma.

Transcriptome studies are revealing the complex gene expression basis of limb regeneration in the primary salamander model - Ambystoma mexicanum (axolotl). To better understand this complexity, there is need to extend analyses to additional salamander species. Using microarray and RNA-Seq, we performed a comparative transcriptomic study using A. mexicanum and two other ambystomatid salamanders: A. andersoni, and A. maculatum. Salamanders were administered forelimb amputations and RNA was isolated and analyzed to identify 405 non-redundant genes that were commonly, differentially expressed 24 h post amputation. Many of the upregulated genes are predicted to function in wound healing and developmental processes, while many of the downregulated genes are typically expressed in muscle. The conserved transcriptional changes identified in this study provide a high-confidence dataset for identifying factors that simultaneous orchestrate wound healing and regeneration processes in response to injury, and more generally for identifying genes that are essential for salamander limb regeneration.

A Tale of Two Axolotls.

The Mexican axolotl (Ambystoma mexicanum) is an icon of culture, a revered aquarium pet, and a highly valued animal model in biomedical research. Unfortunately, Mexican axolotls are critically endangered in their natural Xochimilco habitat in Mexico City. If axolotls go extinct, current efforts to conserve the Xochimilico ecosystem will be undermined, as will efforts to genetically manage the laboratory populations that are needed to sustain research efforts around the world. A concerted global effort is needed to protect and manage this irreplaceable species in natural and laboratory environments.

Flatworm Transcriptomes Reveal Widespread Parasitism by Histophagous Ciliates.

Unicellular ciliates like Tetrahymena are best known as free-living bacteriovores, but many species are facultative or obligate parasites. These "histophages" feed on the tissues of hosts ranging from planarian flatworms to commercially important fish and the larvae of imperiled freshwater mussels. Here, we developed a novel bioinformatics pipeline incorporating the nonstandard ciliate genetic code and used it to search for Ciliophora sequences in 34 publicly available Platyhelminthes EST libraries. From 2,615,036 screened ESTs, we identified nearly 6,000 high-confidence ciliate transcripts, supporting parasitism of seven additional flatworm species. We also cultured and identified Tetrahymena from nine terrestrial and freshwater planarians, including invasive earthworm predators from the genus Bipalium and the widely studied regeneration models Dugesia japonica and Schmidtea mediterranea. A co-phylogenetic reconstruction provides strong evidence for the coevolution of histophagous Ciliophora with their Platyhelminthes hosts. We further report the antiprotozoal aminoglycoside paromomycin expels Tetrahymena from S. mediterranea, providing new opportunities to investigate the effects of this relationship on planarian biology. Together, our findings raise the possibility that invasive flatworms constitute a novel dispersal mechanism for Tetrahymena parasites and position the Platyhelminthes as an ideal model phylum for studying the ecology and evolution of histophagous ciliates.

FLATWORM TRANSCRIPTOMES REVEAL WIDESPREAD PARASITISM BY HISTOPHAGOUS CILIATES.

Unicellular ciliates like Tetrahymena are best known as free-living bacteriovores, but many species are facultative or obligate parasites. These 'histophages' feed on the tissues of hosts ranging from planarian flatworms to commercially important fish and the larvae of imperiled freshwater mussels. Here, we developed a novel bioinformatics pipeline incorporating the nonstandard ciliate genetic code and used it to search for Ciliophora sequences in 34 publicly available Platyhelminthes EST libraries. From 2,615,036 screened ESTs, we identified nearly 6,000 high-confidence ciliate transcripts, supporting parasitism of seven additional flatworm species. We also cultured and identified Tetrahymena from nine terrestrial and freshwater planarians, including invasive earthworm predators from the genus Bipalium and the widely studied regeneration models Dugesia japonica and Schmidtea mediterranea. A cophylogenetic reconstruction provides strong evidence for coevolution of histophagous Ciliophora with their Platyhelminthes hosts. We further report the antiprotozoal aminoglycoside paromomycin expels Tetrahymena from S. mediterranea, providing new opportunities to investigate the effects of this relationship on planarian biology. Together, our findings raise the possibility that invasive flatworms constitute a novel dispersal mechanism for Tetrahymena parasites and position the Platyhelminthes as an ideal model phylum for studying the ecology and evolution of histophagous ciliates.

Nested hierarchal organization of conservation for microRNAs and their putative targets to Drosophila melanogaster.

This study examined microRNA network properties traced through taxonomic hierarchy considering both the acquisition of potential network targets and regulators. Primary literature review and database analyses were conducted to establish modules of conserved microRNAs across metazoan taxonomy. A hierarchical schema for the conservation of microRNAs and their putative targets to Drosophila melanogaster was engineered through comprehensive meta-analysis, and conservation history of 90.39% of the total Drosophila dataset could be resolved through this hierarchical sampling regime; tracing from taxonomic order down to empire. The findings presented in this study represent a documentation of Drosophila microRNA regulatory network behavior thorough taxonomic hierarchy. MicroRNA regulatory network properties were found to transect taxonomic hierarchy. Newly acquired microRNAs from novel families reinforce the pre-existing regulatory network, while expanding the target list to include a small number of novel genes. Lineage specific microRNAs were found to exhibit far fewer conserved targets than do the more broadly conserved microRNAs; even when considering only more recently emerged targets. There was a dramatic expansion in network complexity with the expansion of the microRNA repertoire, and this corresponds to the expansion in biological complexity.

A de novo reference transcriptome for Bolitoglossa vallecula, an Andean mountain salamander in Colombia.

The amphibian order Caudata, contains several important model species for biological research. However, there is need to generate transcriptome data from representative species of the primary salamander families. Here we describe a de novo reference transcriptome for a terrestrial salamander, Bolitoglossa vallecula (Caudata: Plethodontidae). We employed paired-end (PE) illumina RNA sequencing to assemble a de novo reference transcriptome for B. vallecula. Assembled transcripts were compared against sequences from other vertebrate taxa to identify orthologous genes, and compared to the transcriptome of a close plethodontid relative (Bolitoglossa ramosi) to identify commonly expressed genes in the skin. This dataset should be useful to future comparative studies aimed at understanding important biological process, such as immunity, wound healing, and the production of antimicrobial compounds.

HDAC Regulates Transcription at the Outset of Axolotl Tail Regeneration.

Tissue regeneration is associated with complex changes in gene expression and post-translational modifications of proteins, including transcription factors and histones that comprise chromatin. We tested 172 compounds designed to target epigenetic mechanisms in an axolotl (Ambystoma mexicanum) embryo tail regeneration assay. A relatively large number of compounds (N = 55) inhibited tail regeneration, including 18 histone deacetylase inhibitors (HDACi). In particular, romidepsin, an FDA-approved anticancer drug, potently inhibited tail regeneration when embryos were treated continuously for 7 days. Additional experiments revealed that romidepsin acted within a very narrow, post-injury window. Romidepsin treatment for only 1-minute post amputation inhibited regeneration through the first 7 days, however after this time, regeneration commenced with variable outgrowth of tailfin tissue and abnormal patterning. Microarray analysis showed that romidepsin altered early, transcriptional responses at 3 and 6-hour post-amputation, especially targeting genes that are implicated in tumor cell death, as well as genes that function in the regulation of transcription, cell differentiation, cell proliferation, pattern specification, and tissue morphogenesis. Our results show that HDAC activity is required at the time of tail amputation to regulate the initial transcriptional response to injury and regeneration.

Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

Does use of isotretinoin rule out a career in flying?

AIM: To evaluate whether previous isotretinoin use induces permanent, measurable, and clinically significant abnormalities in night vision such that flying is precluded, and whether potential military and civilian commercial aviators should be screened routinely. METHODS: A retrospective, non-interventional, consecutive case series of 47 individuals with a confirmed history of oral isotretinoin use were compared to 20 age and sex matched controls. RESULTS: 47 individuals (44 males and three females), age range 17-33, underwent Goldmann-Weekers dark adaptation (DA) and standard electroretinogram (ERG) according to ISCEV protocols. 34 patients showed no abnormality in any parameters. Two patients had abnormal DA and ERGs. The mean scotopic ERG b wave amplitude of the isotretinoin group was 496.5 microV (SD 51.3 microV) compared with 501.7 microV (62.3.1 microV) among the controls. The group mean a:b ratio was 0.55 (0.04) compared to 0.69 (0.08) in the controls. CONCLUSION: Previous use of isotretinoin may have caused retinal toxicity in two subjects and laboratory evidence of night blindness in 11 further subjects. One subject had subclinical changes remaining in the ERG 96 months after cessation of isotretinoin. This may justify the directed use of electrophysiological screening in professions that are night vision critical.

DNA damage responses at low radiation doses.

Increased cell killing after exposure to low acute doses of X rays (0-0.5 Gy) has been demonstrated in cells of a number of human tumor cell lines. The mechanisms underlying this effect have been assumed to be related to a threshold dose above which DNA repair efficiency or fidelity increases. We have used cells of two radioresistant human tumor cell lines, one that shows increased sensitivity to low radiation doses (T98G) and one that does not (U373), to investigate the DNA damage response at low doses in detail and to establish whether there is a discontinuous dose response or threshold in activation of any important mediators of this response. In the two cell lines studied, we found a sensitive, linear dose response in early signaling and transduction pathways between doses of 0.1 and 2 Gy with no evidence of a threshold dose. We demonstrate that ATM-dependent signaling events to downstream targets including TP53, CHK1 and CHK2 occur after doses as low as 0.2 Gy and that these events promote an effective damage response. Using chemical inhibition of specific DNA repair enzymes, we show that inhibition of DNA-PK-dependent end joining has relatively little effect at low (<1 Gy) doses in hyper-radiosensitive cells and that at these doses the influence of RAD51-mediated repair events may increase, based on high levels of RAD51/BRCA2 repair foci. These data do not support a threshold model for activation of DNA repair in hyper-radiosensitive cells but do suggest that the balance of repair enzyme activity may change at low doses.

Transcriptomics using axolotls.

Microarray and RNA-sequencing technology now exists for the characterization of the Ambystoma mexicanum transcriptome. With sufficient replication, these tools give the opportunity to truly investigate gene expression in a variety of experimental paradigms. Analysis of data from the Amby002 array and RNA-sequencing technology can identify genes that change expression levels in concert with each other, which in turn may reveal mechanisms associated with biological processes and molecular functions.

Sal-Site: research resources for the Mexican axolotl.

Sal-Site serves axolotl research efforts by providing Web access to genomic data and information, and living stocks that are reared and made available by the Ambystoma Genetic Stock Center (AGSC). In this chapter, we detail how investigators can search for genes of interest among Sal-Site resources to identify orthologous nucleotide and protein-coding sequences, determine genome positions within the Ambystoma meiotic map, and obtain estimates of gene expression. In the near future, additional genomic resources will be made available for the axolotl, including a listing of genes that are partially or wholly contained within Bacterial Artificial Chromosome (BAC) vectors, a prioritized collection of deeply sequenced BAC clones, chromosome-specific assemblies of genomic DNA, and transgenic axolotls that are engineered using TALENs and CRISPRs. Also, services provided by the AGSC will be expanded to include microinjection of user constructs into single cell embryos and distribution of axolotl tissues, DNA, and RNA. In conclusion, Sal-Site is a useful resource that generates, shares, and evolves Ambystoma associated information and databases to serve research and education.

Flavone acetic acid induced changes in human endothelial permeability: potentiation by tumour-conditioned medium.

Flavone acetic acid (FAA) causes significant regression of larger established tumours in murine in vivo systems. This in vivo effect of FAA has been shown to include a vascular component. In an effort to elucidate the mechanism of action of FAA, we have studied the effects of FAA on the permeability of human endothelium in vitro. Monolayers of human umbilical vein endothelial cells (HUVEC) grown on polycarbonate filters were incubated in 1 mg/ml FAA for 120 min at 37 degrees C. During the first 60 min, there was a 6-8-fold increase in permeability; this was followed by a return to control levels even in the continued presence of FAA. In contrast, in the presence of tumour conditioned medium, FAA caused a rapid 6-fold increase in permeability which did not subsequently return to control levels. The permeability changes which occurred under the latter conditions were accompanied by a rapid contraction of the cytoskeleton. The permeability of monolayers of human melanoma cells was unaffected by FAA.

A comparison of colorimetric and clonogenic assays for hypoxic-specific toxins with hamster and human cells.

The hypoxic cytotoxicities of misonidazole and pimonidazole (Ro 03-8799) towards the human tumor cell lines HT-1080 and LoVo have been compared with those seen with Chinese hamster V79-379A cells. Survival was assayed using two colorimetric assays, either a tetrazolium salt (MTT) or methylene blue, and by conventional colony scoring. The drugs were more cytotoxic towards HT-1080 and LoVo cells than V79 cells. The times taken for 10 mmol dm-3 misonidazole to reduce survival to 0.1 surviving fraction (SF) using colony formation as the end point were 2.6 hr for HT-1080, 2.4 hr for LoVo, and 3.5 hr for V79; using the MTT assay these times were 3.5 hr, 2.1 hr, and 2.9 hr, respectively. The times for 2 mmol dm-3 pimonidazole to reduce survival to 0.1 SF using colony formation as the end point were 2.0 hr for HT-1080, 1.7 hr for LoVo, and 3.7 hr for V79; using the MTT assay these times were 2.5 hr, 1.4 hr, and 2.5 hr, respectively.

Flavone acetic acid as a modifier of endothelial cell function.

Flavone acetic acid (FAA) causes significant regression of larger established tumors in murine systems in vivo, but is only slightly toxic in vitro. This in vivo effect is thought to be indirect, or immunological, rather than a direct cytotoxic effect on tumor cells. Using the WHFIB fibrosarcoma, which grows both in vivo and in vitro, and the murine endothelial cell line B10, we have studied the effect of FAA on the survival of tumor and endothelial cells in vitro. The times taken for 1 mg ml-1 FAA to reduce survival to 0.1 surviving fraction were 63 hr for B10 and greater than 85 hr for WHFIB in vitro. WHFIB tumors in vivo were more sensitive than tumor cells in vitro, a single dose of 150 mg kg-1 FAA inducing a tumor growth delay of 10 days at treatment size + 2 mm. As FAA is more toxic to tumor-bearing animals than to those which are non-tumor bearing the effect of tumor conditioned medium on the cytotoxicity of FAA toward B10 cells was studied; no enhanced effect was seen. As FAA is only weakly cytotoxic in vitro to endothelial cells, and even less so to tumor cells, sublethal effects of FAA on endothelial cell function in vitro were studied. The permeability of monolayers of human unbilical vein endothelial cells (HUVEC) in vitro is transiently increased by FAA. Also, procoagulant activity of HUVEC is induced by FAA and this activity is further enhanced in the presence of a factor isolated from Meth-A tumor cells.

Radiosensitizer-DNA interactions in relation to intracellular uptake.

We have studied the intracellular uptake of a number of neutral, acidic, and basic radiosensitizers. For neutral sensitizers, we observed a correlation between the measured intracellular concentration and sensitization, but for bases, a large change in average intracellular concentration results in only a small change in sensitization. In addition, by modifying the intralysosomal pH, we have altered the measured average intracellular concentration of the weak base pimonidazole by a factor of two, although this had no detectable effect upon sensitization. Using spin filtration of solutions of sensitizers with naked calf thymus DNA or chromatin we have assessed the affinity of DNA for sensitizers with different prototropic and lipophilic properties. We have also shown that this anomalous behavior of the basic sensitizers could be partly explained on the basis of intracellular localization adjacent to the DNA due to ionic interactions. Thus, intracellular localization needs to be considered when interpreting average intracellular uptake data.

Synthesis and activity of novel nitropyrazines for use as hypoxic cell radiosensitizers.

A series of eight novel nitropyrazines has been prepared by oxidation of sulfoximine intermediates. The partition coefficient, one-electron reduction potential, sensitizer enhancement ratio, and chronic and acute aerobic cytotoxicity have been measured for each. Two representatives of this series were tested in the Ames test and were not found to be mutagenic.

Effects of novel and conventional anti-cancer agents on human endothelial permeability: influence of tumour secreted factors.

A number of anti-cancer agents have been implicated in vascular toxicity. The effects have been attributed to direct drug toxicity towards endothelium. Little attention has been focussed on the interaction between anticancer drugs, endothelial cells and tumour secreted factors. It is well known that tumours can secrete factors such as vascular permeability factor which do affect endothelial cells and could alter their response to the vascular effects of anticancer drugs. In the present study, we have examined, in vitro, the direct effects of vinblastine (VBL), 5-fluorouracil (5-FU), melphalan (L-PAM) and the novel tubulin inhibitor combretastatin A-1 (CBS) on endothelial permeability under normal and tumour simulated conditions. Monolayers of human umbilical vein endothelial cells (HUVEC) grown on membrane filters were incubated in drug in normal growth medium or medium conditioned by the human melanoma cell line, RPMI-7951 (TCM). VBL caused a rapid increase in permeability during the first 20 minutes, which was maintained for the duration of the experiment (120 minutes). The effect was not altered by TCM or restored to control levels when VBL was replaced by drug-free medium. Similarly, CBS caused a rapid increase in permeability; however, in contrast to VBL, this increase was enhanced by TCM. The changes induced by VBL and CBS were accompanied by contraction of the endothelial F-actin cytoskeleton. Neither L-PAM nor 5-FU altered the permeability of HUVEC monolayers. This study demonstrates that certain anti-cancer agents have a direct effect on endothelial cells, leading to an increase in the permeability of endothelial monolayers. Both VBL and CBS have vascular components in their mode of action which may lead to vascular collapse and tumour necrosis.

A single-shot rapid-mixing device for radiobiological studies with mammalian cells.

A single-shot rapid-mixing device is described for the rapid addition of solutions of radiation-modifying agents, to cell suspensions, at well-defined times relative to a pulse of radiation. The liquid injection system could be used to initiate or quench a wide range of chemical or biochemical reactions. The rapid-mixing device is based on a syringe driven by a stepper motor and can inject up to 2 cm3 liquid in less than 100 ms. The radiation source, a 4 MV Van de Graaff accelerator, provides an electron beam which is deflected from the beam dump on to the sample in two stages, providing a 10 ms radiation pulse. A digital delay circuit defines the interval between mixing and irradiation. The apparatus has been designed to study the kinetics of processes that occur over a time range extending from about 0.1 s to some minutes. It bridges the gap between the ranges available with conventional fast-mixing and those using standard X- or gamma-irradiation methods. The time resolution of the technique has been examined by following the timecourse of radiosensitization by oxygen in mammalian cells. The timecourse of radioprotection of aerobic mammalian cells by dithiothreitol has been measured using the technique.

Low dose hypersensitivity in the T98G human glioblastoma cell line.

PURPOSE: To examine the low dose-response of a human radioresistant glioblastoma cell line (T98G) using two different methods to measure surviving fraction and to define the influence of cell cycle phase on this response. MATERIALS AND METHODS: The survival of cells following exposure to single very low doses of X-rays in vitro was measured using either the Dynamic Microscopic Image Processing Scanner (DMIPS) or a Cell Sorter (CS). The DMIPS was also used to measure the low dose survival response of T98G cells following manipulation of their progression through the cell cycle. RESULTS: With both methods, T98G demonstrated marked low dose hyper-radiosensitivity (HRS) and the two methods produced very similar data in the low dose region of the survival curve. However, the CS protocol produced less variable results and was the more efficient method of generating low dose data. HRS was also demonstrated when these cells were irradiated while held in reversible arrest in the G1 phase of the cell cycle, but the effect was less marked than in the asynchronous population. CONCLUSIONS: T98G glioblastoma cells demonstrate marked HRS, which is a characteristic of the whole population rather than being due to the influence of a small subpopulation of hyper-radiosensitive cells within a particular phase of the cell cycle.