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1.
Genome Biol Evol ; 16(2)2024 02 01.
Article in English | MEDLINE | ID: mdl-38242711

ABSTRACT

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.


Subject(s)
Ciliophora , Planarians , Animals , Phylogeny , Transcriptome , Ciliophora/genetics , Planarians/genetics
2.
bioRxiv ; 2023 Sep 17.
Article in English | MEDLINE | ID: mdl-37745455

ABSTRACT

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.

3.
Data Brief ; 29: 105256, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32123704

ABSTRACT

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.

4.
Sci Rep ; 9(1): 6751, 2019 05 01.
Article in English | MEDLINE | ID: mdl-31043677

ABSTRACT

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.


Subject(s)
Ambystoma mexicanum/physiology , Histone Deacetylases/metabolism , Regeneration , Tail/physiology , Transcription, Genetic , Animals , Computational Biology/methods , Epigenomics , Gene Expression Profiling , Gene Expression Regulation/drug effects , Histone Deacetylase Inhibitors/pharmacology , Regeneration/drug effects
5.
Genomics ; 111(6): 1216-1225, 2019 12.
Article in English | MEDLINE | ID: mdl-30092345

ABSTRACT

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.


Subject(s)
Regeneration/genetics , Transcriptome , Ambystoma , Ambystoma mexicanum , Animals , Extremities/physiology , Gene Expression Profiling , Gene Ontology
6.
Sci Rep ; 7(1): 6, 2017 01 31.
Article in English | MEDLINE | ID: mdl-28127056

ABSTRACT

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.


Subject(s)
Ambystoma mexicanum/genetics , Biological Variation, Population , Genotype , Pigments, Biological/genetics , Animals , Biological Evolution , DNA/genetics
7.
Methods Mol Biol ; 1290: 309-19, 2015.
Article in English | MEDLINE | ID: mdl-25740496

ABSTRACT

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.


Subject(s)
Ambystoma mexicanum/genetics , Gene Expression Profiling/methods , Animals , Cluster Analysis , Molecular Sequence Annotation , Oligonucleotide Array Sequence Analysis , RNA/genetics , RNA/isolation & purification , Sequence Analysis, RNA
8.
Methods Mol Biol ; 1290: 321-36, 2015.
Article in English | MEDLINE | ID: mdl-25740497

ABSTRACT

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.


Subject(s)
Ambystoma mexicanum , Computational Biology/methods , Internet , Ambystoma mexicanum/genetics , Animals , Databases, Genetic , Expressed Sequence Tags/metabolism , Gene Expression Profiling , Genetic Markers/genetics , Genomics , User-Computer Interface
9.
Bioscience ; 65(12): 1134-1140, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-32123398

ABSTRACT

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.

10.
Chem Biodivers ; 9(5): 945-64, 2012 May.
Article in English | MEDLINE | ID: mdl-22589095

ABSTRACT

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.


Subject(s)
MicroRNAs/genetics , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Regulatory Networks/genetics , MicroRNAs/classification , MicroRNAs/metabolism , Phylogeny
11.
Eye (Lond) ; 22(1): 18-25, 2008 Jan.
Article in English | MEDLINE | ID: mdl-16778823

ABSTRACT

AIMS: To analyse outcomes, factors influencing surgical success, and surgical technique of Molteno implantation over the past 11 years in order to identify ways of improving long-term control. METHODS: Retrospective interventional review of case records of all consecutive patients undergoing Molteno implantation at Groote Schuur Hospital between 1/1/1991 and 31/12/2002. Data were recorded on an MSAccess database and processed using Kaplan-Meier survival curves and life table analysis. RESULTS: We analysed 162 consecutive single-phase Molteno tube implantation procedures on 157 eyes of 148 patients with mean follow-up of 2.9 years. Intraocular pressure (IOP) dropped from a mean of 43.3 at booking to 19.1 at final follow-up. Overall 'complete success' was achieved in 30% and 'partial success' in 16%. A high preoperative IOP was a significant predictor of a high postoperative pressure. Pseudophakic patients had significantly better postoperative pressure control. Neovascular glaucoma was a risk factor for poor pressure control. Race, gender, previous surgery, uveitis, and trauma did not influence surgical outcome. Follow-up adjusted incidence of 2.4 cases of endophthalmitis per patient year was unexpectedly high. Tubes that migrated had been secured with absorbable sutures in 4/5 cases. CONCLUSIONS: In this study, high preoperative IOPs were probably a significant contributing factor to relatively poor postoperative pressure control. Addressing this issue may aid in improving outcomes in future surgery. The high postoperative pressure outcomes suggest that single plate Molteno implantation is not an ideal way of achieving low target pressure in third world glaucoma patients.


Subject(s)
Glaucoma/surgery , Intraocular Pressure/physiology , Molteno Implants/standards , Female , Humans , Male , Prosthesis Design/standards , Retrospective Studies , South Africa , Statistics as Topic , Treatment Outcome , Visual Acuity
12.
Br J Ophthalmol ; 90(8): 957-9, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16723361

ABSTRACT

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.


Subject(s)
Aerospace Medicine , Isotretinoin/adverse effects , Keratolytic Agents/adverse effects , Vision Disorders/chemically induced , Adolescent , Adult , Career Choice , Dark Adaptation/drug effects , Electroretinography , Female , Humans , Male , Night Blindness/chemically induced , Occupational Health , Personnel Selection , Retinal Diseases/chemically induced , Retrospective Studies
13.
Radiat Res ; 164(3): 292-302, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16137202

ABSTRACT

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.


Subject(s)
Cell Cycle Proteins/metabolism , Cell Survival/radiation effects , DNA Damage , DNA, Neoplasm/radiation effects , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/radiation effects , Glioma/metabolism , Neoplasm Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins , Cell Line, Tumor , Differential Threshold/radiation effects , Dose-Response Relationship, Radiation , Glioma/pathology , Humans , Radiation Dosage , Signal Transduction/radiation effects
14.
Poult Sci ; 83(12): 1940-3, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15615003

ABSTRACT

The purpose of this study was to determine if playing hen calls at the feeder affects broiler chick productivity and welfare. Hatched chicks (n = 832) were equally placed into 16 pens. Broilers in 8 treated pens received 3 min of hen-feeding calls once each hour during the first 9 d of age; broilers in the other 8 pens received no recorded hen vocalizations during the same period. After d 9, recorded hen vocalizations ceased and all birds were treated identically. Through 9 d of age, chicks receiving recorded hen vocalizations had improved (P < or = 0.05) feed conversion ratios, and these chicks weighed more (139.12+/-1.52 g vs. 133.17+/-1.59 g for control chicks; P < or = 0.01). The behavior data showed that on d 1, 4, and 7, more (P < or = 0.05) chicks receiving recorded hen vocalizations were found within 0.61 m of the speaker than control chicks. Following recorded hen vocalization cessation on d 9, birds and feed were weighed on 17, 24, 31, and 38 d of age, and carcass yield was measured on d 40. There were no differences in BW, feed efficiency, or carcass yield after recorded hen vocalization ceased. These data suggest that after d 9, differences became nonsignificant, corresponding to when recorded hen vocalization stopped. Behavior data demonstrated that chicks appear to be attracted when stimulated with recorded hen vocalizations, thus remaining in close proximity to the speaker. Evidence suggests that hen vocalization improves production and attraction to hen vocalization with known improvements in BW and feed conversion during the first 9 d posthatch.


Subject(s)
Chickens/physiology , Feeding Behavior/physiology , Vocalization, Animal/physiology , Animal Husbandry , Animals , Body Weight , Female , Male , Tape Recording
16.
Int J Radiat Biol ; 79(2): 99-105, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12569013

ABSTRACT

PURPOSE: To examine the low-dose radiation response of human glioma cell lines separated into different cell-cycle phases and to determine if low-dose hyper-radiosensitivity (HRS) differs in populations defined by cell-cycle position. To assess whether predictions of the outcome of multiple low-dose regimens should take account of cell-cycle effects. MATERIALS AND METHODS: The clonogenic survival of G1, G2 and S phase cells was measured after exposure to single doses of X-rays in two human glioma cell lines. One cell line (T98G) showed marked HRS when asynchronous cells were irradiated, while the other (U373) did not. Separation of populations and high-resolution cell counting was achieved using a fluorescence activated cell sorter. Sorted cell populations were irradiated with 240 kVp X-rays to doses between 0.05 and 5Gy. The resulting cell-survival versus dose data were comparatively fitted using the linear-quadratic and induced-repair models in order to assess the degree of HRS. RESULTS: In both cell lines the low-dose response was altered when different populations were irradiated. In T98G cells, all populations showed HRS, but this was most marked in G2 phase cells. In U373 cells, no HRS was found in G1 or S phase cells, but HRS was demonstrable in G2 phase cells. CONCLUSIONS: HRS was expressed by the whole cell population of T98G cells but the size of the effect varied with cell-cycle phase and was most marked in the G2 population. In U373 cells, the effect could only be demonstrated in G2 cells. This implies that HRS is primarily a response of G2 phase cells and that this response dominates that seen in asynchronous populations. Actively proliferating cell populations may therefore demonstrate a greater increase in radiosensitivity to very low radiation doses compared with quiescent populations.


Subject(s)
Cell Cycle/radiation effects , Glioblastoma/pathology , Glioblastoma/radiotherapy , Cell Survival/radiation effects , Dose-Response Relationship, Radiation , Humans , Models, Biological , Radiation Tolerance , Tumor Cells, Cultured
17.
Brain Dev ; 23 Suppl 1: S44-9, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11738841

ABSTRACT

Development and retention of speech is reported in 265 people with Rett syndrome: 30% (80) never gained real words, 55% (145) gained real words and lost them, 15%(40) retained some words and 6% of the total (16/265) continued to use phrases appropriately. Morphological studies of the cytoarchitecture of the speech areas in 14 cases indicate the existence of interhemispheric differences which form part of the infrastructure for speech processing. Ten adults with Rett syndrome and with meaningful speech are compared to age matched adults without speech. The profile of mind and strategies for coping with its problems are described by a family. Although the range in severity is wide the mental profile is remarkably consistent across the severity range with regard to both positive and negative aspects.


Subject(s)
Rett Syndrome/genetics , Rett Syndrome/psychology , Speech Disorders/epidemiology , Speech Disorders/genetics , Adolescent , Adult , Brain/growth & development , Brain/pathology , Brain/physiopathology , Child , Child, Preschool , Disability Evaluation , Dosage Compensation, Genetic , Female , Humans , Mutation/genetics , Rett Syndrome/pathology , Speech Disorders/pathology
18.
Int J Radiat Biol ; 77(6): 655-64, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11403705

ABSTRACT

PURPOSE: It was demonstrated previously that some radioresistant tumour cell lines respond to decreasing single, low radiation doses by becoming increasingly radiosensitive. This paper reports the response of four radioresistant human glioma cell lines to multiple low-dose radiation exposures given at various intervals. Three of the cell lines (T98G, U87, A7) were proven already to show low-dose hyper-radiosensitivity (HRS) after single low doses; the fourth, U373, does not show HRS after acute doses. MATERIALS AND METHODS: Clonogenic cell-survival measurements were made in vitro using the Dynamic Microscopic Image Processing Scanner (DMIPS) or Cell Sorter (CS) following exposure to 240kVp X-rays one or more times. RESULTS: A consistent, time-dependent hypersensitive response to a second, or subsequent, dose was observed in the cell lines that demonstrated HRS. This time-dependent change in radiosensitivity did not occur in the radioresistant cell line that did not show HRS (U373). In one cell line that demonstrated strong HRS, T98G, a similar time-dependent hypersensitive response was also seen when the cells were irradiated whilst held in the G1-phase of the cell cycle. In this same cell line, significantly increased cell kill was demonstrated when three very low doses (0.4 Gy) were given per day, 4 h apart, for 5 days, compared with the same total dose given as once-daily 1.2Gy fractions. CONCLUSIONS: These data demonstrate the possibility that a multipledose per day, low-dose per fraction regimen, termed 'ultrafractionation', could produce increased tumour cell kill in radioresistant tumours compared with the same total dose given as conventional-sized 2 Gy fractions.


Subject(s)
Glioma/radiotherapy , Astrocytoma/pathology , Astrocytoma/radiotherapy , Cell Survival/radiation effects , Dose-Response Relationship, Radiation , G1 Phase , Glioblastoma/pathology , Glioblastoma/radiotherapy , Glioma/pathology , Humans , Radiation Tolerance , Tumor Cells, Cultured , Tumor Stem Cell Assay
19.
Phys Med Biol ; 46(1): 63-76, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11197679

ABSTRACT

Investigating the effect of low-dose radiation exposure on cells using assays of colony-forming ability requires large cell samples to maintain statistical accuracy. Manually counting the resulting colonies is a laborious task in which consistent objectivity is hard to achieve. This is true especially with some mammalian cell lines which form poorly defined or 'fuzzy' colonies, typified by glioma or fibroblast cell lines. A computer-vision-based automated colony counter is presented in this paper. It utilizes novel imaging and image-processing methods involving a modified form of the Hough transform. The automated counter is able to identify less-discrete cell colonies typical of these cell lines. The results of automated colony counting are compared with those from four manual (human) colony counts for the cell lines HT29, A172, U118 and IN1265. The results from the automated counts fall well within the distribution of the manual counts for all four cell lines with respect to surviving fraction (SF) versus dose curves, SF values at 2 Gy (SF2) and total area under the SF curve (Dbar). From the variation in the counts, it is shown that the automated counts are generally more consistent than the manual counts.


Subject(s)
Automation , Cell Count , Cells, Cultured/radiation effects , Cytological Techniques/methods , Cell Line , Cell Separation , Cell Survival , Dose-Response Relationship, Radiation , Fibroblasts/cytology , Flow Cytometry , Humans , Image Processing, Computer-Assisted/methods , Reproducibility of Results , Software , Tumor Cells, Cultured
20.
Int J Radiat Biol ; 75(11): 1341-8, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10597908

ABSTRACT

PURPOSE: To examine the low-dose radiation response of a series of radioresistant human glioma cell lines and determine if low-dose hypersensitivity is a characteristic of these cells. MATERIALS AND METHODS: The clonogenic survival of six radioresistant human glioma cell lines was measured following exposure to graded, single, very low doses of X-rays in vitro. High resolution was achieved using either a Dynamic Microscopic Image Processing Scanner (DMIPS) or a cell sorter (CS). RESULTS: In five of the six cell lines tested, low-dose hypersensitivity (HRS) was demonstrated although in the sixth, a grade III astrocytoma line, it was not. These results are consistent with previous data indicating that low-dose hypersensitivity is more marked in more radioresistant cell lines although the difference between the glioblastoma cell lines with differing SF2 is not marked. CONCLUSION: Low-dose hypersensitivity is common in radioresistant glioma cell lines. This may have implications for the treatment of these tumours if further studies confirm that HRS translates to increased effectiveness per gray in vivo when very low doses per fraction are used.


Subject(s)
Astrocytoma/pathology , Glioblastoma/pathology , Tumor Cells, Cultured/radiation effects , Astrocytoma/radiotherapy , Cell Separation , Cell Survival/radiation effects , Dose-Response Relationship, Radiation , Flow Cytometry , Glioblastoma/radiotherapy , Humans , Linear Models , Radiation Tolerance , Tumor Cells, Cultured/pathology
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