The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

• MeSH
• DNA genetika   MeSH
• eukaryotické buňky MeSH
• kometový test metody   MeSH
• lidé MeSH
• poškození DNA * MeSH
• pyrimidinové dimery * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA MeSH
• pyrimidinové dimery * MeSH

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

• Klíčová slova
• Calibration, Comet assay, Radiation, Ring-trial, Standardisation,
• MeSH
• ionizující záření * MeSH
• kalibrace MeSH
• kometový test metody   MeSH
• lidé MeSH
• poškození DNA * MeSH
• savci MeSH
• vztah dávky záření a odpovědi MeSH
• záření gama MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND AND OBJECTIVES: To investigate the genetic determinants of the most common type of antibody-mediated autoimmune encephalitis, anti-NMDA receptor (anti-NMDAR) encephalitis. METHODS: We performed a genome-wide association study in 178 patients with anti-NMDAR encephalitis and 590 healthy controls, followed by a colocalization analysis to identify putatively causal genes. RESULTS: We identified 2 independent risk loci harboring genome-wide significant variants (p < 5 × 10-8, OR ≥ 2.2), 1 on chromosome 15, harboring only the LRRK1 gene, and 1 on chromosome 11 centered on the ACP2 and NR1H3 genes in a larger region of high linkage disequilibrium. Colocalization signals with expression quantitative trait loci for different brain regions and immune cell types suggested ACP2, NR1H3, MADD, DDB2, and C11orf49 as putatively causal genes. The best candidate genes in each region are LRRK1, encoding leucine-rich repeat kinase 1, a protein involved in B-cell development, and NR1H3 liver X receptor alpha, a transcription factor whose activation inhibits inflammatory processes. DISCUSSION: This study provides evidence for relevant genetic determinants of antibody-mediated autoimmune encephalitides outside the human leukocyte antigen (HLA) region. The results suggest that future studies with larger sample sizes will successfully identify additional genetic determinants and contribute to the elucidation of the pathomechanism.

• MeSH
• celogenomová asociační studie * MeSH
• dospělí MeSH
• encefalitida s protilátkami proti NMDA receptorům genetika   MeSH
• genetické lokusy MeSH
• lidé MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2'-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2'-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 μL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo, but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease.

• Klíčová slova
• Biomarkers, DNA, DNA repair, Nucleotide pool, Oxidative stress, RNA,
• MeSH
• 8-hydroxy-2'-deoxyguanosin MeSH
• biologické markery MeSH
• chromatografie kapalinová MeSH
• deoxyguanosin MeSH
• lidé MeSH
• nukleové kyseliny * MeSH
• oxidační stres MeSH
• poškození DNA MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• 8-hydroxy-2'-deoxyguanosin MeSH
• biologické markery MeSH
• deoxyguanosin MeSH
• nukleové kyseliny * MeSH

The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations.

• Klíčová slova
• Biomarkers, Comet assay, DNA damage, Human biomonitoring, Pooled analysis,
• MeSH
• biologické markery krev   MeSH
• kometový test metody   MeSH
• lidé MeSH
• poškození DNA genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• biologické markery MeSH

This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.

• MeSH
• buněčné linie MeSH
• kometový test metody   MeSH
• lidé MeSH
• oprava DNA * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.

• MeSH
• dodržování směrnic statistika a číselné údaje   MeSH
• kometový test metody   normy   MeSH
• konsensus MeSH
• laboratoře MeSH
• lidé MeSH
• výzkumný projekt * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

We previously demonstrated beneficial effects of 22 h of hyperoxia following near-lethal porcine hemorrhagic shock, whereas therapeutic hypothermia was detrimental. Therefore, we investigated whether shorter exposure to hyperoxia (12 h) would still improve organ function, and whether 12 h of hypothermia with subsequent rewarming could avoid deleterious effects after less severe hemorrhagic shock.Twenty-seven anesthetized and surgically instrumented pigs underwent 3 h of hemorrhagic shock by removal of 30% of the blood volume and titration of the mean arterial blood pressure (MAP) to 40 mm Hg. Post-shock, pigs were randomly assigned to control, hyperoxia (FIO2 100% for 12 h) or hypothermia group (34°C core temperature for 12 h with subsequent rewarming). Before, at the end of shock, after 12 and 23 h of resuscitation, data sets comprising hemodynamics, blood gases, and parameters of inflammation and organ function were acquired. Postmortem, kidney samples were collected for immunohistochemistry and western blotting.Hyperoxia exerted neither beneficial nor detrimental effects. In contrast, mortality in the hypothermia group was significantly higher compared with controls (67% vs. 11%). Hypothermia impaired circulation (MAP 64 (57;89) mm Hg vs. 104 (98; 114) mm Hg) resulting in metabolic acidosis (lactate 11.0 (6.6;13.6) mmol L vs. 1.0 (0.8;1.5) mmol L) and reduced creatinine clearance (26 (9;61) mL min vs. 77 (52;80) mL min) compared to the control group after 12 h of resuscitation. Impaired kidney function coincided with increased renal 3-nitrotyrosine formation and extravascular albumin accumulation.In conclusion, hyperoxia proved to be safe during resuscitation from hemorrhagic shock. The lacking organ-protective effects of hyperoxia compared to resuscitation from near-lethal hemorrhage suggest a dependence of the effectiveness of hyperoxia from shock severity. In line with our previous report, therapeutic hypothermia (and rewarming) was confirmed to be detrimental most likely due to vascular barrier dysfunction.

• MeSH
• analýza krevních plynů MeSH
• hemodynamika fyziologie   MeSH
• hemoragický šok metabolismus   terapie   MeSH
• hyperoxie metabolismus   terapie   MeSH
• oxidační stres fyziologie   MeSH
• prasata MeSH
• synthasa oxidu dusnatého, typ II metabolismus   MeSH
• terapeutická hypotermie MeSH
• tyrosin analogy a deriváty   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-nitrotyrosine MeSH Prohlížeč
• synthasa oxidu dusnatého, typ II MeSH
• tyrosin MeSH

OBJECTIVE: Hemorrhagic shock-induced tissue hypoxia induces hyperinflammation, ultimately causing multiple organ failure. Hyperoxia and hypothermia can attenuate tissue hypoxia due to increased oxygen supply and decreased demand, respectively. Therefore, we tested the hypothesis whether mild therapeutic hypothermia and hyperoxia would attenuate postshock hyperinflammation and thereby organ dysfunction. DESIGN: Prospective, controlled, randomized study. SETTING: University animal research laboratory. SUBJECTS: Thirty-six Bretoncelles-Meishan-Willebrand pigs of either gender. INTERVENTIONS: After 4 hours of hemorrhagic shock (removal of 30% of the blood volume, subsequent titration of mean arterial pressure at 35 mm Hg), anesthetized and instrumented pigs were randomly assigned to "control" (standard resuscitation: retransfusion of shed blood, fluid resuscitation, norepinephrine titrated to maintain mean arterial pressure at preshock values, mechanical ventilation titrated to maintain arterial oxygen saturation > 90%), "hyperoxia" (standard resuscitation, but FIO2, 1.0), "hypothermia" (standard resuscitation, but core temperature 34°C), or "combi" (hyperoxia plus hypothermia) (n = 9 each). MEASUREMENTS AND MAIN RESULTS: Before, immediately at the end of and 12 and 22 hours after hemorrhagic shock, we measured hemodynamics, blood gases, acid-base status, metabolism, organ function, cytokine production, and coagulation. Postmortem kidney specimen were taken for histological evaluation, immunohistochemistry (nitrotyrosine, cystathionine γ-lyase, activated caspase-3, and extravascular albumin), and immunoblotting (nuclear factor-κB, hypoxia-inducible factor-1α, heme oxygenase-1, inducible nitric oxide synthase, B-cell lymphoma-extra large, and protein expression of the endogenous nuclear factor-κB inhibitor). Although hyperoxia alone attenuated the postshock hyperinflammation and thereby tended to improve visceral organ function, hypothermia and combi treatment had no beneficial effect. CONCLUSIONS: During resuscitation from near-lethal hemorrhagic shock, hyperoxia attenuated hyperinflammation, and thereby showed a favorable trend toward improved organ function. The lacking efficacy of hypothermia was most likely due to more pronounced barrier dysfunction with vascular leakage-induced circulatory failure.

• MeSH
• analýza krevních plynů MeSH
• cytokiny metabolismus   MeSH
• hemodynamika MeSH
• hemokoagulace fyziologie   MeSH
• hemoragický šok patofyziologie   terapie   MeSH
• hyperoxie * MeSH
• imunoblotting MeSH
• imunohistochemie MeSH
• ledviny patologie   MeSH
• náhodné rozdělení MeSH
• prasata MeSH
• prospektivní studie MeSH
• resuscitace metody   MeSH
• tekutinová terapie MeSH
• terapeutická hypotermie metody   MeSH
• umělé dýchání MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH

The concentration of the estrogens 17beta-estradiol, estriol, estrone, 17alpha-ethinylestradiol, mestranol and norethisterone and of the anthropogenic gadolinium (Gd(ant)) has been determined in the creeks and rivers, sewage treatment plants and water works of the city of Prague. The rapid degradation of estrogens in surface water allows the estrogen concentration gradient to be used as a very precise and sensitive guideline by which to pin-point sewage leaks into surface run-off water. The rather conservative behavior of Gd(ant) in surface and ground water documents in the present case the presence of sewage water in the surface water cycle.

• MeSH
• chemické látky znečišťující vodu analýza   MeSH
• estrogeny analýza   MeSH
• gadolinium analýza   MeSH
• odpadní vody chemie   MeSH
• senzitivita a specificita MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• chemické látky znečišťující vodu MeSH
• estrogeny MeSH
• gadolinium MeSH
• odpadní vody MeSH