| 1. |
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| 2. |
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| 3. |
- Castillejo-Lopez, Casimiro, et al.
(författare)
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Genetic and physical interaction of the B-cell systemic lupus erythematosus-associated genes BANK1 and BLK
- 2012
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 71:1, s. 136-142
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Tidskriftsartikel (refereegranskat)abstract
- ObjectivesAltered signalling in B cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signalling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterise the role of BANK1 and BLK in SLE, a genetic interaction analysis was performed hypothesising that genetic interactions could reveal functional pathways relevant to disease pathogenesis.MethodsThe GPAT16 method was used to analyse the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localisation, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK.ResultsEpistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK. As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, the possibility that BANK1 and BLK could also show a protein-protein interaction was tested. The co-immunoprecipitation and co-localisation of BLK and BANK1 were demonstrated. In a Daudi cell line and primary naive B cells endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies.ConclusionsThis study shows a genetic interaction between BANK1 and BLK, and demonstrates that these molecules interact physically. The results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signalling pathway.
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| 4. |
- Kulka, U., et al.
(författare)
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Realising the European Network of Biodosimetry (RENEB)
- 2012
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Ingår i: Radiation Protection Dosimetry. - : Oxford University Press (OUP). - 0144-8420 .- 1742-3406. ; 151:4, s. 621-625
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Tidskriftsartikel (refereegranskat)abstract
- In Europe, a network for biological dosimetry has been created to strengthen the emergency preparedness and response capabilities in case of a large-scale nuclear accident or radiological emergency. Through the RENEB (Realising the European Network of Biodosimetry) project, 23 experienced laboratories from 16 European countries will establish a sustainable network for rapid, comprehensive and standardised biodosimetry provision that would be urgently required in an emergency situation on European ground. The foundation of the network is formed by five main pillars: (1) the ad hoc operational basis, (2) a basis of future developments, (3) an effective quality-management system, (4) arrangements to guarantee long-term sustainability and (5) awareness of the existence of RENEB. RENEB will thus provide a mechanism for quick, efficient and reliable support within the European radiation emergency management. The scientific basis of RENEB will concurrently contribute to increased safety in the field of radiation protection.
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| 5. |
- Zdzalik, M., et al.
(författare)
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Prevalence of genes encoding extracellular proteases in Staphylococcus aureus - important targets triggering immune response in vivo
- 2012
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Ingår i: Fems Immunology and Medical Microbiology. - 0928-8244. ; 66:2, s. 220-229
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Tidskriftsartikel (refereegranskat)abstract
- Proteases of Staphylococcus aureus have long been considered to function as important virulence factors, although direct evidence of the role of particular enzymes remains incomplete and elusive. Here, we sought to provide a collective view of the prevalence of extracellular protease genes in genomes of commensal and pathogenic strains of S.aureus and their expression in the course of human and mouse infection. Data on V8 protease, staphopains A and B, aureolysin, and the recently described and poorly characterized group of six Spl proteases are provided. A phylogenetically diverse collection of 167 clinical isolates was analyzed, resulting in the comprehensive genetic survey of the prevalence of protease-encoding genes. No correlation between identified gene patterns with specific infections was established. Humoral response against the proteases of interest was examined in the sera derived from human patients and from a model mouse infection. The analysis suggests that at least some, if not all, tested proteases are expressed and secreted during the course of infection. Overall, the results presented in this study support the hypothesis that the secretory proteases as a group may contribute to the virulence of S.aureus.
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| 6. |
- Ainsbury, E A, et al.
(författare)
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REVIEW OF RETROSPECTIVE DOSIMETRY TECHNIQUES FOR EXTERNAL IONISING RADIATION EXPOSURES.
- 2011
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Ingår i: Radiation Protection Dosimetry. - : Oxford University Press (OUP). - 0144-8420 .- 1742-3406. ; 147:4, s. 573-592
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Tidskriftsartikel (refereegranskat)abstract
- The current focus on networking and mutual assistance in the management of radiation accidents or incidents has demonstrated the importance of a joined-up approach in physical and biological dosimetry. To this end, the European Radiation Dosimetry Working Group 10 on 'Retrospective Dosimetry' has been set up by individuals from a wide range of disciplines across Europe. Here, established and emerging dosimetry methods are reviewed, which can be used immediately and retrospectively following external ionising radiation exposure. Endpoints and assays include dicentrics, translocations, premature chromosome condensation, micronuclei, somatic mutations, gene expression, electron paramagnetic resonance, thermoluminescence, optically stimulated luminescence, neutron activation, haematology, protein biomarkers and analytical dose reconstruction. Individual characteristics of these techniques, their limitations and potential for further development are reviewed, and their usefulness in specific exposure scenarios is discussed. Whilst no single technique fulfils the criteria of an ideal dosemeter, an integrated approach using multiple techniques tailored to the exposure scenario can cover most requirements.
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| 7. |
- Beltrán-Pardo, Eliana, et al.
(författare)
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Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development
- 2013
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Ingår i: PLOS ONE. - 1932-6203. ; 8:9, s. e72098-
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Tidskriftsartikel (refereegranskat)abstract
- Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies havedocumented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differentialeffects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effectof gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from theeutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, andlate stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses,while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival withhigher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might havetaken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and latestage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that thetolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this patternare unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of responsemechanisms to damaged DNA in the course of development.
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| 8. |
- Dulski, J., et al.
(författare)
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Neuroacanthocytosis - Clinical variability (a report on six cases)
- 2014
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Ingår i: Movement Disorders. - : Wiley. - 0885-3185. ; 29:Suppl 1, s. 194-194
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Konferensbidrag (refereegranskat)abstract
- Objective: To provide clinical clues to differential diagnosis in patients with chorea and other movement disorders with blood acanthocytes. Background: Neuroacanthocytosis (NA) is an umbrella term for neurological conditions associated with acanthocytosis. Core NA syndromes, with basal ganglia involvement and in which acanthocytosis is a frequent finding, include autosomal recessive choreaacanthocytosis (Ch-Ac) and X-linked McLeod syndrome (MLS). Due to the very low prevalence, scarcity of data and high clinical variability they may be underdiagnosed. Methods: Six male patients (pts), three diagnosed with Ch-Ac: 33-y.o.(no.1), 35-y.o.(no.2), 42-y.o.(no.3), two diagnosed with MLS: 52-y.o.(no.4), 60-y.o.(no.5) and one 62-y.o.(no.6), a brother of no.5, with clinical suspicion of MLS. The patients had an unremarkable family history and were asymptomatic until adulthood. Pts no.1,2,4,5,6 developed generalized chorea and patient no.3 had predominant bradykinesia. Pts no.1,2,3 had phonic and motor tics, additionally pts no.1 and 2 exhibited peculiar oromandibular dystonia with tongue thrusting. In pts no.2 and 3 dystonic supination of feet was observed, patient no.3 subsequently developed bilateral foot drop. Pts no. 2 and 4 had signs of muscle atrophy. Tendon reflexes were decreased or absent and electroneurography demonstrated sensorimotor neuropathy in patients no. 1,2,3,4,5, except no. 6. Generalized seizures were seen in patients no.2,3,5,6 and myoclonic jerks in patient no 1. Cognitive deterioration was reported in patients no.1,2,3,5,6. Serum creatine kinase levels were elevated in all six patients. Results: Peripheral blood smears revealed acanthocytosis in patients no.1,2,3,5,6, except no. 4. In patients no. 1 and 3 reduced expression of chorein was detected on Western blot. In patient no. 2 genetic testing showed mutations in VPS13A gene and in no.4 and 5 genetic analysis confirmed mutations in XK gene (MLS). The time from the onset of symptoms till establishing the diagnosis in patients no. 1,2,3,4,5 was 11,5,7,6,32 years respectively. Patient no.4 suddenly developed multiple organ failure and died of cardiac arrhythmia at the age of 52. Conclusions: We highlight the variability of clinical presentation of NA syndromes and the long time from the onset to diagnosis with the need to screen the blood smears in uncertain cases, however, as in one of our cases acanthocytes may even be not found.
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| 9. |
- Martinelli-Boneschi, F, et al.
(författare)
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A genome-wide association study in progressive multiple sclerosis
- 2012
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Ingår i: Multiple sclerosis (Houndmills, Basingstoke, England). - : SAGE Publications. - 1477-0970 .- 1352-4585. ; 18:10, s. 1384-1394
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Tidskriftsartikel (refereegranskat)abstract
- Background: The role played by genetic factors in influencing the clinical course of multiple sclerosis (MS) is not yet well established. Objective: We aimed to identify genetic variants associated with progressive MS (PrMS). Methods: We conducted a genome-wide association study (GWAS) in 197 patients with PrMS and 234 controls of Italian origin. We tested the top 20 single nucleotide polymorphisms (SNPs) with suggestive evidence of association ( p-value<10−4) in two independent sets of primary progressive MS cases and controls. Results: We identified a risk-associated SNP in the HLA region in linkage disequilibrium (LD) with DRB1*1501 and DQB*0602 loci, with genome-wide significance (rs3129934T, pcombined=6.7×10-16, OR=2.34, 95% CI=1.90–2.87), and a novel locus on chromosome 7q35 with suggestive evidence of association (rs996343G, pcombined=2.4×10-5, OR=0.70, 95% CI=0.59–0.83) which maps within a human endogenous retroviral (HERV) element. The new locus did not have a ‘ cis’ effect on RNA expression in lymphoblastic cell lines, but pathway analyses of ‘ trans’ effects point to an expression regulation of genes involved in neurodegeneration, including glutamate metabolism ( p<0.01) and axonal guidance signalling ( p<0.02). Conclusions: We have confirmed the established association with the HLA region and, despite the low statistical power of the study, we found suggestive evidence for association with a novel locus on chromosome 7, with a putative regulatory role.
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| 10. |
- Beltran-Pardo, Eliana, et al.
(författare)
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Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development
- 2013
-
Ingår i: PLoS ONE. - : Public Library of Science. - 1932-6203. ; 8:9
-
Tidskriftsartikel (refereegranskat)abstract
- Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies havedocumented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differentialeffects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effectof gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from theeutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, andlate stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses,while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival withhigher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might havetaken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and latestage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that thetolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this patternare unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of responsemechanisms to damaged DNA in the course of development.
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| 11. |
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| 12. |
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| 13. |
- Borowski, Tomasz, et al.
(författare)
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The alkenyl migration mechanism catalyzed by extradiol dioxygenases : a hybrid dft study
- 2012
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 17:6, s. 881-890
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Tidskriftsartikel (refereegranskat)abstract
- 6-Hydroxymethyl-6-methylcyclohexa-2,4-dienone is a mechanistic probe which when incubated with an extradiol dioxygenase yields a 2-tropolone product. This observation was originally interpreted as evidence supporting a direct heterolytic 1,2-alkenyl migration mechanism for a ring expansion reaction catalyzed by this class of Fe(II)-dependent nonheme enzymes (Xin and Bugg in J Am Chem Soc 130:10422-10430, 2008). In the work reported in this contribution we used quantum chemical methods to test whether such a mechanism is energetically possible and we found that it is not, neither for the mechanistic probe nor for the native catalytic cycle intermediate. Models of increasing complexity were used to calculate energy barriers to the heterolytic 1,2-alkenyl migration and alternative radical mechanisms. It was found that the former involves substantially higher barriers than the latter. A tentative radical mechanism that accounts for the transformation of the probe substrate to 2-tropolone was also proposed, and it involves acceptable barriers.
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| 14. |
- Deperas-Kaminska, Marta, et al.
(författare)
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Inter-chromosomal variation in aberration frequencies in human lymphocytes exposed to charged particles of LET between 0.5 and 55 keV/mu m
- 2010
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 86:11, s. 975-985
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: To investigate the distribution of chromosomal aberrations in chromosomes 2, 8 and 14 induced by charged particles, using the fluorescence in situ hybridisation (FISH) technique. Methods: Irradiation of peripheral blood from six healthy volunteers (four male and two female) was performed at the accelerators of the Joint Institute for Nuclear Research (JINR) in Dubna (Russia). Whole blood samples were irradiated with 2 and 3 Gy of protons (170 MeV/nucleon (n), linear energy transfer (LET) approximate to 0.5 keV/mu m), 3.5 Gy of C-12 ions (480 MeV/n, LET = 10.6 keV/mu m), 3 Gy of C-12 ions 500 MeV/n, LET = 12 keV/mu m), 4 Gy of Li-7 ions (30 MeV/n, LET approximate to 20 keV/mu m) and 3 Gy of B-11 ions (32 MeV/n, LET approximate to 55 keV/mu m). Chromosomal aberrations were analysed in metaphase and prematurely condensed chromosomes (PCC) induced in G(2)-cells using calyculin A. Chromosomes 2, 8 and 14 were painted in different colours and aberrations scored with the help of an image-analysis system. Results: Chromosome 2 was generally less sensitive than expected on the basis of its DNA content. A higher than expected frequency of exchanges was found in chromosomes 8 and 14. On average, the dicentric frequency in chromosome 2 was higher than the translocation frequency, whereas variable dicentric to translocation ratios were observed in chromosomes 8 and 14. When aberrations in all painted chromosomes were summed up the ratio was close to 1. The frequency of complex aberrations correlated with LET. Conclusion: In lymphocytes of donors studied in this work chromosome 2 appears to be consistently less sensitive to protons and heavy ions than chromosomes 8 and 14. Complex aberrations appear to be a potential marker of radiation quality.
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| 15. |
- Herman, Jeremy S., et al.
(författare)
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Land-Bridge Calibration of Molecular Clocks and the Post-Glacial Colonization of Scandinavia by the Eurasian Field Vole Microtus agrestis
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 9:8, s. e103949-
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Tidskriftsartikel (refereegranskat)abstract
- Phylogeography interprets molecular genetic variation in a spatial and temporal context. Molecular clocks are frequently used to calibrate phylogeographic analyses, however there is mounting evidence that molecular rates decay over the relevant timescales. It is therefore essential that an appropriate rate is determined, consistent with the temporal scale of the specific analysis. This can be achieved by using temporally spaced data such as ancient DNA or by relating the divergence of lineages directly to contemporaneous external events of known time. Here we calibrate a Eurasian field vole ( Microtus agrestis) mitochondrial genealogy from the well-established series of post-glacial geophysical changes that led to the formation of the Baltic Sea and the separation of the Scandinavian peninsula from the central European mainland. The field vole exhibits the common phylogeographic pattern of Scandinavian colonization from both the north and the south, however the southernmost of the two relevant lineages appears to have originated in situ on the Scandinavian peninsula, or possibly in the adjacent island of Zealand, around the close of the Younger Dryas. The mitochondrial substitution rate and the timescale for the genealogy are closely consistent with those obtained with a previous calibration, based on the separation of the British Isles from mainland Europe. However the result here is arguably more certain, given the level of confidence that can be placed in one of the central assumptions of the calibration, that field voles could not survive the last glaciation of the southern part of the Scandinavian peninsula. Furthermore, the similarity between the molecular clock rate estimated here and those obtained by sampling heterochronous (ancient) DNA ( including that of a congeneric species) suggest that there is little disparity between the measured genetic divergence and the population divergence that is implicit in our land-bridge calibration.
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| 16. |
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| 17. |
- Romm, H., et al.
(författare)
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Automatic scoring of dicentric chromosomes as a tool in large scale radiation accidents
- 2013
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Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis. - : Elsevier BV. - 1383-5718 .- 1879-3592. ; 756:1-2, s. 174-183
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Tidskriftsartikel (refereegranskat)abstract
- Mass casualty scenarios of radiation exposure require high throughput biological dosimetry techniques for population triage in order to rapidly identify individuals who require clinical treatment. The manual dicentric assay is a highly suitable technique, but it is also very time consuming and requires well trained scorers. In the framework of the MULTIBIODOSE EU FP7 project, semi-automated dicentric scoring has been established in six European biodosimetry laboratories. Whole blood was irradiated with a Co-60 gamma source resulting in 8 different doses between 0 and 4.5 Gy and then shipped to the six participating laboratories. To investigate two different scoring strategies, cell cultures were set up with short term (2-3 h) or long term (24 h) colcemid treatment. Three classifiers for automatic dicentric detection were applied, two of which were developed specifically for these two different culture techniques. The automation procedure included metaphase finding, capture of cells at high resolution and detection of dicentric candidates. The automatically detected dicentric candidates were then evaluated by a trained human scorer, which led to the term 'semi-automated' being applied to the analysis. The six participating laboratories established at least one semi-automated calibration curve each, using the appropriate classifier for their colcemid treatment time. There was no significant difference between the calibration curves established, regardless of the classifier used. The ratio of false positive to true positive dicentric candidates was dose dependent. The total staff effort required for analysing 150 metaphases using the semi-automated approach was 2 min as opposed to 60 min for manual scoring of 50 metaphases. Semi-automated dicentric scoring is a useful tool in a large scale radiation accident as it enables high throughput screening of samples for fast triage of potentially exposed individuals. Furthermore, the results from the participating laboratories were comparable which supports networking between laboratories for this assay.
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| 18. |
- Romm, H., et al.
(författare)
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VALIDATION OF SEMI-AUTOMATIC SCORING OF DICENTRIC CHROMOSOMES AFTER SIMULATION OF THREE DIFFERENT IRRADIATION SCENARIOS
- 2014
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Ingår i: Health Physics. - 0017-9078 .- 1538-5159. ; 106:6, s. 764-771
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Tidskriftsartikel (refereegranskat)abstract
- Large scale radiological emergencies require high throughput techniques of biological dosimetry for population triage in order to identify individuals indicated for medical treatment. The dicentric assay is the gold standard technique for the performance of biological dosimetry, but it is very time consuming and needs well trained scorers. To increase the throughput of blood samples, semi-automation of dicentric scoring was investigated in the framework of the MULTIBIODOSE EU FP7 project, and dose effect curves were established in six biodosimetry laboratories. To validate these dose effect curves, blood samples from 33 healthy donors (>10 donors/scenario) were irradiated in vitro with Co-60 gamma rays simulating three different exposure scenarios: acute whole body, partial body, and protracted exposure, with three different doses for each scenario. All the blood samples were irradiated at Ghent University, Belgium, and then shipped blind coded to the participating laboratories. The blood samples were set up by each lab using their own standard protocols, and metaphase slides were prepared to validate the calibration curves established by semi-automatic dicentric scoring. In order to achieve this, 300 metaphases per sample were captured, and the doses were estimated using the newly formed dose effect curves. After acute uniform exposure, all laboratories were able to distinguish between 0 Gy, 0.5 Gy, 2.0, and 4.0 Gy (p < 0.001), and, in most cases, the dose estimates were within a range of +/- 0.5 Gy of the given dose. After protracted exposure, all laboratories were able to distinguish between 1.0 Gy, 2.0 Gy, and 4.0 Gy (p < 0.001), and here also a large number of the dose estimates were within +/- 0.5 Gy of the irradiation dose. After simulated partial body exposure, all laboratories were able to distinguish between 2.0 Gy, 4.0 Gy, and 6.0 Gy (p < 0.001). Overdispersion of the dicentric distribution enabled the detection of the partial body samples; however, this result was clearly dose-dependent. For partial body exposures, only a few dose estimates were in the range of +/- 0.5 Gy of the given dose, but an improvement could be achieved with higher cell numbers. The new method of semi-automation of the dicentric assay was introduced successfully in a network of six laboratories. It is therefore concluded that this method can be used as a high-throughput screening tool in a large-scale radiation accident.
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| 19. |
- Salomaa, Sisko, et al.
(författare)
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State of the art in research into the risk of low dose radiation exposure-findings of the fourth MELODI workshop
- 2013
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Ingår i: Journal of Radiological Protection. - : IOP Publishing. - 0952-4746 .- 1361-6498. ; 33:3, s. 589-603
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Tidskriftsartikel (refereegranskat)abstract
- The fourth workshop of the Multidisciplinary European Low Dose Initiative (MELODI) was organised by STUK-Radiation and Nuclear Safety Authority of Finland. It took place from 12 to 14 September 2012 in Helsinki, Finland. The meeting was attended by 179 scientists and professionals engaged in radiation research and radiation protection. We summarise the major scientific findings of the workshop and the recommendations for updating the MELODI Strategic Research Agenda and Road Map for future low dose research activities.
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| 20. |
- Wojcik, Anna, et al.
(författare)
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Mechanism of Benzylic Hydroxylation by 4-Hydroxymandelate Synthase : A Computational Study
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:47, s. 9570-9580
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Tidskriftsartikel (refereegranskat)abstract
- Hydroxymandelate synthase (HMS) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) are highly related enzymes using the same substrates but catalyzing hydroxylation reactions yielding different products. The first The first steps of the HMS and I-IPPD catalytic reactions are believed to proceed in the same way and lead to an Fe(IV)=O-hydroxyphenylacetate (HPA) intermediate. Further, down the, catalytic cycles, HMS uses Fe(IV)=O to perform hydroxylation of the benzylic carbon, Whereas in HPPD, the reactive oxoferryl intermediate attacks the aromatic ring of HPA. This study focuses on this part of the HMS catalytic cycle that starts from the oxoferryl intermediate and aims to identify interactions within the active site that are responsible for enzyme specificity. To this end, a HMS-Fe(IV)=O-HPA complex was modeled with molecular dynamics simulations On the basis. of the molecular: dynamics. equilibrated structure, active site model suitable for quantum chemical Investigations was constructed and used for density functional theory. (B3LYP) Calculations of the mechanism of the native reaction of HMS, i.e., benzylic hydroxylation, and the alternative electrophilic attack on the ring, which is a step Of the HPPD catalytic cycle: The most important, result of this study is the finding that the conformation of the Ser201 side chain in the second coordination shell has a key role in directing the of Fe(IV)=O. into either the HMS or the HPPD channel
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| 21. |
- Wojcik, Anna, et al.
(författare)
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Role of Substrate Positioning in the Catalytic Reaction of 4-Hydroxyphenylpyruvate Dioxygenase-A QM/MM Study
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 136:41, s. 14472-14485
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Tidskriftsartikel (refereegranskat)abstract
- Ring hydroxylation and coupled rearrangement reactions catalyzed by 4-hydroxyphenylpyruvate dioxygenase were studied with the QM/MM method ONIOM(B3LYP:AMBER). For electrophilic attack of the ferryl species on the aromatic ring, five channels were considered: attacks on the three ring atoms closest to the oxo ligand (C1, C2, C6) and insertion of oxygen across two bonds formed by them (C1-C2, C1-C6). For the subsequent migration of the carboxymethyl substituent, two possible directions were tested (C1-C2, C1-C6), and two different mechanisms were sought (stepwise radical, single-step heterolytic). In addition, formation of an epoxide (side)product and benzylic hydroxylation, as catalyzed by the closely related hydroxymandelate synthase, were investigated. From the computed reaction free energy profiles it follows that the most likely mechanism of 4-hydroxyphenylpyruvate dioxygenase involves electrophilic attack on the C1 carbon of the ring and subsequent single-step heterolytic migration of the substituent. Computed values of the kinetic isotope effect for this step are inverse, consistent with available experimental data. Electronic structure arguments for the preferred mechanism of attack on the ring are also presented.
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