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PubMed1999

The comet-tail artifact: an ultrasound sign ruling out pneumothorax.

OBJECTIVE: Ultrasound artifacts arising from the lung-wall interface are either vertical (comet-tail artifacts) or horizontal. The significance of these artifacts for the diagnosis of pneumothorax was assessed. DESIGN: Prospective clinical study. SETTING: The medical ICU of a university-affiliated teaching hospital. PATIENTS: We compared 41 complete pneumothoraces with 146 hemithoraces in 73 critically ill patients in which computed tomography showed absence of pneumothorax. MEASUREMENTS: The anterior chest wall was investigated in supine patients using a portable device. The test was defined as positive for complete pneumothorax when only horizontal artifacts were visible, and negative when artifacts arising from the pleural line and spreading up to the edge of the screen (referred to as "comet-tail artifacts") were present. RESULTS: The feasibility was 98%. Ultrasound showed exclusive horizontal artifacts in all 41 analyzable cases of complete pneumothorax. In the pneumothorax-free group, "comet-tail artifacts" were present in 87 cases and exclusive horizontal artifacts in 56. Ultrasound as well as computed tomography showed anterior consolidation or anterior pleural effusion in three cases. Horizontal artifacts had a sensitivity and a negative predictive value of 100% and a specificity of 60% for the diagnosis of pneumothorax. Horizontal artifacts and absent lung sliding, when combined, had a sensitivity and a negative predictive value of 100% and a specificity of 96.5%. CONCLUSIONS: Ultrasound detection of the "comet-tail artifact" at the anterior chest wall allows complete pneumothorax to be discounted.

Adolescent↗
PubMed1999

Early detection of staurosporine-induced apoptosis by comet and annexin V assays.

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction.

Animals↗
PubMed2004

How should COMET influence heart failure practice?

Much clinical experience has led us to assume that the administration of a b-blocker, regardless of dose or frequency, would produce similar mortality benefits in patients with heart failure. The results from the recently published Carvedilol or Metoprolol European Trial (COMET), which found greater benefit of carvedilol than immediate-release metoprolol on mortality, clearly demonstrated this is not true. In heart failure, the COMET results strongly support the use of beta-blockers that have proven effective in large-scale clinical trials. The primary disagreement regarding COMET concerns the explanation of the efficacy difference between the two b-blockers tested. Pharmacodynamic considerations and hemodynamic data from the COMET trial itself suggest there were unequal degrees of b1-blockade between patients receiving carvedilol and immediate-release metoprolol. Failure to achieve a similar degree of b1-receptor blockade in the two groups prevents conclusions regarding the potential incremental benefits of selective versus nonselective adrenergic blockade. Further studies are needed to determine whether there are additional clinical benefits from the inhibition of adrenergic receptors beyond the proven benefits of b1-blockade.

Adrenergic alpha-Antagonists↗
PubMed1987

Comets and life.

Some of the chemical species which have been detected in comets include H2O, HCN, CH3CN, CO, CO2, NH3, CS, C2 and C3. All of these have also been detected in the interstellar medium, indicating a probable relationship between interstellar dust and gas clouds and comets. Laboratory experiments carried out with different mixtures of these molecules give rise to the formation of the biochemical compounds which are necessary for life, such as amino acids, purines, pyrimidines, monosaccharides, etc. However, in spite of suggestions to the contrary, the presence of life in comets is unlikely. On the other hand, the capture of cometary matter by the primitive Earth is considered essential for the development of life on this planet. The amount of cometary carbon-containing matter captured by the Earth, as calculated by different authors, is several times larger than the total amount of organic matter present in the biosphere (10(18)g). The major classes of reactions which were probably involved in the formation of key biochemical compounds are discussed. Our tentative conclusions are that: 1) comets played a predominant role in the emergence of life on our planet, and 2) they are the cosmic connection with extraterrestrial life.

Atmosphere↗
PubMed2006

Ultrasound lung comets: a clinically useful sign of extravascular lung water.

Assessment of extravascular lung water is a challenging task for the clinical cardiologist and an elusive target for the echocardiographer. Today chest x-ray is considered the best way to assess extravascular lung water objectively, but this requires radiology facilities and specific reading expertise, uses ionizing energy, and poses a significant logistic burden. Recently, a new method was developed using echocardiography (with cardiac probes) of the lung. An increase in extravascular lung water-as assessed independently by chest computed tomography, chest x-ray, and thermodilution techniques-is mirrored by appearance of ultrasound lung comets (ULCs). ULCs consist of multiple comet tails originating from water-thickened interlobular septa and fanning out from the lung surface. The technique requires ultrasound scanning of the anterior right and left chest, from the second to the fifth intercostal space. It is simple (with a learning curve of < 10 examinations) and fast to perform (requiring < 3 minutes). ULC assessment is independent of the cardiac acoustic window, because the lung on the anterior chest is scanned. It requires very basic 2-D technology imaging, even without a second harmonic or Doppler. ULCs probably represent an ultrasonic equivalent of radiologic Kerley B-lines. On still-frame assessment, cardiogenic watery comets can be difficult to distinguish from pneumogenic fibrotic comets, although the latter are usually more localized and are not dissolved by an acute diuretic challenge. Functionally, ULCs are a sign of distress of the alveolar-capillary membrane, often associated with reduced ejection fraction and increased pulmonary wedge pressure. The ULC sign is quantitative, reproducible, and ideally suited to complement conventional echocardiography in the evaluation of heart failure patients in the emergency department (for the differential diagnosis of dyspnea), in-hospital evaluation (for tailoring diuretic therapy), home care (with portable ultrasound), and stress echocardiography lab (as a sign of acute pulmonary congestion during stress). In conclusion, ULCs represent a useful, practical, and appealingly simple way to image directly extravascular lung water.

Artifacts↗
PubMed1997

Comet assay studies on the activation of two diaziridinylbenzoquinones in K562 cells.

Two versions of the comet assay have been used to identify the difference in the modes of action of AZQ (2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone) and BZQ (2,5-diaziridinyl-3,6-bis(ethanolamino)-1,4-benzoquinone) in human leukaemia K562 cells and a K562-derived resistant cell line, BZQR. Using the standard alkaline assay, AZQ produced dose-dependent changes in the mean comet moments from K562 cells, consistent with the formation of strand breaks. This damage was repaired over a period of 6 hr after removal of the drug. The resistant cell line, BZQR, showed much smaller changes in comet moment under identical conditions. In contrast to AZQ, BZQ did not produce any measurable strand breaks in the K562 or BZQR cells. However, the comet radiation/crosslinking assay and a fluorescence-based assay revealed that BZQ extensively cross-links DNA in K562 cells. The extent of cross-linking is greatly reduced in the resistant cell line.

Antineoplastic Agents↗
PubMed2001

A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes.

OBJECTIVE: To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears. DESIGN: The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined. SETTING: Clinical and academic research environment. PATIENT(S): 59 patients undergoing fertility treatment. INTERVENTION(S): Sperm samples leftover from IVF procedures were fixed and processed for the comet assay. MAIN OUTCOME MEASURE(S): Sperm head DNA density and sperm variables. RESULT(S): A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology. CONCLUSION(S): The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.

Acridine Orange↗
PubMed1997

DNA effects in repair-deficient V79 Chinese hamster cells studied with the comet assay.

Using the alkaline comet assay (single cell gel electrophoresis), we studied the induction and persistence of DNA damage induced by methyl methanesulfonate (MMS) and neocarzinostatin (NCS) in the repair-deficient Chinese hamster cell lines V-E5 and XR-V15B. Effects in the comet assay were analyzed directly after treatment as well as after a postincubation period in mutagen-free medium to gain insight into the DNA repair capacities of the mutant cell lines in relation to different primary DNA lesions. Both mutagens caused a concentration-related increase in DNA strand breakage in both mutant cell lines and in the normal parental cell lines. Repair of MMS-induced DNA damage during postincubation was similar in normal and mutant cell lines, while diminished repair was seen after NCS treatment in XR-V15B cells. Our data show that XR-V15B cells only repaired about 30% of NCS-induced DNA damage within 1 h, while the parental V79 cell line repaired about 70%. Since this cell line is defective in the repair of DNA double-strand breaks (DSB), the results indicate that NCS-induced DSB significantly contribute to the genotoxic effects seen in the comet assay. However, compared to previously studied induction of gene mutations and chromosome aberrations, detection of NCS-induced DNA effects with the comet assay was less sensitive and increased DNA migration only occurred under strong cytotoxic conditions.

Animals↗
PubMed1998

Evaluation of the comet assay as a method for the detection of DNA damage in the cells of a marine invertebrate, Mytilus edulis L. (Mollusca: Pelecypoda).

The potential application of the comet assay for monitoring the effect of DNA damaging agents on the marine mussel, Mytilus edulis (an important pollution indicator organism), was explored. A detailed investigation of the baseline levels of single-strand breaks in isolated gill cells, and how they were affected by age/size of animal, time since collection, feeding regime, in vivo vs. in vitro exposure conditions, and by antioxidant supplementation was undertaken. The level of cometing in untreated controls was found to be highly variable over time (fluctuations between low and very high DNA damage occurred over just 14 days post collection). No difference was observed between age/size and feeding regime of the animals. On exposure to 0, 100, 500 and 1000 microM H2O2, it was observed that the in vitro exposure produced a markedly more homogeneous dose response compared to the in vivo studies (where gill cells were exposed as a tissue). An important finding of our research was the effect of prior supplementation of the animals' diet with 1 mg/ml alpha-tocopherol acetate (vitamin E compound), which resulted in a marked reduction in the levels of DNA damage expressed by the negative controls, without influencing the actual response to H2O2 (0, 5, 25, and 100 microM) and N-nitrosodimethylamine, NDMA (0, 5, 25, and 100 mM). The effect of vitamin E supplementation was to increase the sensitivity of the comet assay at the lower end of the dose range. This study demonstrated the potential application of the comet assay to the gill cells of the mussel, M. edulis. Although preliminary findings suggest that antioxidant supplementation can improve the sensitivity of the assay by lowering the baseline damage in untreated animals, our conclusion is that the assay has more potential for use in an in vitro context for the screening of agents destined for release or disposal into the marine environment.

Animals↗
PubMed1996

Benzene-induced genotoxicity in mice in vivo detected by the alkaline comet assay: reduction by CYP2E1 inhibition.

The myelotoxic and genotoxic effects of benzene have been related to oxidative DNA damage after metabolism by CYP2E1. Single cell gel electrophoresis (alkaline comet assay) detects DNA damage and may thus be a convenient method for the study of benzene genotoxicity. Benzene exposure to NMRI mice as a single oral gavage at 40, 200 or 450 mg/kg resulted in dose-related DNA damage indicated by an increased comet tail length of peripheral blood lymphocytes and bone marrow nucleated cells sampled 6 h after exposure. After a dose of 40 mg/kg, there was a 1.6-fold increase of 'tail length' in bone marrow nucleated cells in comparison with the control (p < 0.01). There was no significant increase in DNA damage in peripheral blood lymphocytes in the same animals. At 200 mg/kg, the tail length was 4.8-fold and 4.0-fold increased in the two cell types, respectively (p < 0.01). At 450 mg/kg, the tail length was further increased to 5.4-fold and 6.6-fold of the control values, respectively (p < 0.01). Pretreatment with propylene glycol (5 microliters/g b.wt., twice with a 60-min interval), a selective CYP2E1 inhibitor, reduced the increase in the tail length by about half at all doses in both cell types (p < 0.01). By comparing our data with those from genotoxicity studies on benzene using other methods, we conclude that the 'alkaline comet assay' is a sensitive method to detect DNA damage induced by benzene. We also infer that CYP2E1 contributes, at least partly, to the formation of the 'comet'-inducing metabolites in the chosen cell types.

Animals↗
PubMed1997

The influence of cell growth, detoxifying enzymes and DNA repair on hydrogen peroxide-mediated DNA damage (measured using the comet assay) in human cells.

Single-cell gel electrophoresis (the comet assay) is a sensitive method for detecting strand breaks at the level of individual cells. Cells embedded in agarose are lysed, electrophoresed, and fluorescently stained. Breaks in the DNA release its supercoiling and allow DNA to extend toward the anode, resembling a comet. We have used the comet assay to investigate the influence of growth state, xenobiotic detoxifying enzymes, and DNA repair processes on the response of cultured human cells to oxidative damage. HepG2 and Caco-2 cells are differentiated liver and colon cell lines, respectively. HeLa and GM1899A cells are relatively unspecialized epithelial and lymphoblastoid cells. Substrate-dependent cells showed a cyclical fluctuation of glutathione (GSH) with respect to growth. Enzyme activities (glutathione reductase, glutathione peroxidase, and catalase) varied considerably between cell types and changed with cell growth state. Hydrogen peroxide induced more DNA damage in actively dividing cells than in confluent cultures. Sensitivity to oxidative injury did not correlate with detoxifying enzyme activity. Rather, differences in susceptibility between cells could be correlated with differences in DNA repair capacity. This study highlights the need to standardize experimental conditions if the comet assay is to be employed in the study of genotoxicity.

Catalase↗
PubMed1998

In vivo response of mouse liver to gamma-radiation assessed by the comet assay.

The alkaline comet assay was used to measure DNA damage induced in liver cells of mice irradiated with gamma-radiation, as well as the repair competency of these cells. A simplified procedure for the isolation of nuclei from cells in solid tissues was developed. This simplified method allows nuclei to be processed into lysis only 5 min after briefly chilling the tissue to depress any enzymatic activity. The nuclei were spontaneously released by a sharp cut of the tissue and exposure of the cut to a drop of 50 mM sodium-phosphate buffer at pH 7.2, immediately before adding the low melting agarose. Thus, the procedure minimizes time-dependent modification of the endogenous level of damage by reducing additional strand breaks or repair produced during processing. The induction of DNA damage by gamma-radiation behaved as a one-hit event in the liver cells, as there was a positive linear correlation between the radiation dose and the fraction of DNA migrated into the comet tails. The level of DNA damage produced by gamma-radiation was highly significant at doses of 0.5 and 1 Gy. Based on the mean extent of DNA migration, the level of damage was not reduced following only one hour of repair time however, after two hours, there was a significant reduction in DNA migration. To increase the resolution of the statistical analysis, the nuclei of each sample were distributed in five types of comets, according to the percentage of DNA in the tail. To compare the frequency distributions of these types of comets between different experimental situations, a Pearson chi-square statistical analysis was applied. It was found, by this analysis, that the DNA repair which occurred 1 h after 1 Gy of gamma-irradiation is significant and that, after 2 h, more DNA repair occurs, but a significant residual damage still persists when comparing this sample with the control.

Animals↗
PubMed1998

Application of the comet assay for monitoring DNA damage in workers exposed to chronic low-dose irradiation. II. Base damage.

In the preceding paper [M. Wojewodzka, M. Kruszewski, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation. I. Strand breakage., Mutat. Res. 416 (1998) 21-35], we reported the results of DNA damage examination carried out for a group of people (49 individuals) professionally at risk of exposure to low doses of ionizing radiation as measured by the alkaline comet assay. Here, we used the method in combination with oxidative base damage-specific endonucleases to estimate base damage in the same individuals. These were endonuclease III (endoIII) and formamidopyrimidine glycosylase (FPG). In contrast to the previous investigations, we found no statistically significant difference in base damage between the control and hazard groups. Interestingly, the hazard group exhibited lower level of enzyme-sensitive sites than the control; however, this different was not significant. No correlation of base damage with age was found, similarly as in the case of DNA damage measured by the alkaline comet assay. Interindividual variability of base damage precluded exposure estimation for single individuals, since several members of the control group exhibited high comet parameters.

Adult↗
PubMed1998

Genotoxic bifunctional aldehydes produce specific images in the comet assay.

Seven genotoxic aldehydes (acrolein, chloroacetaldehyde, crotonaldehyde, formaldehyde, glutardialdehyde, glyoxal, and methylacrolein) have been studied in vitro using the alkaline version of the comet assay (or single cell microgel electrophoresis assay) in freshly isolated rat hepatocytes. Chloroacetaldehyde, glyoxal and methylacrolein treatment resulted in an elevated tail moment (TM), used as indicator for an DNA damaging activity and formation of comet like structures. In addition, this treatment also caused characteristic DNA spot images with small, highly condensed areas within the otherwise circular DNA spots. These were not seen in solvent and N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated control cells. Treatment of hepatocytes with acrolein, crotonaldehyde, formaldehyde and glutardialdehyde resulted in an TM which did not differ from those of control values although 86-95% of the cells showed characteristic changes of their DNA spot images. The condensed areas are probably the consequence of the known DNA and protein crosslinking activities of these bifunctional aldehydes. It is suggested that using the alkaline comet assay both TM (or overall comet length) as well as changes in the DNA spot image should be evaluated.

Aldehydes↗
PubMed1999

Lack of adverse effect of smoking habit on DNA strand breakage and base damage, as revealed by the alkaline comet assay.

In our preceding papers [M. Wojewódzka, M. Kruszewski, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: I. Strand breakage, Mutat. Res., 416 (1998) 21-35; M. Kruszewski, M. Wojewódzka, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: II. Base damage, Mutat. Res. , 416 (1998) 37-57.], we evaluated the DNA breakage and base damage with the use of comet assay in a group of 49 workers chronically exposed to low doses of ionizing radiation. There was a statistically significant difference in the damage levels between the hazard and control group. In this paper we describe a confounding lack of effect of the smoking habit on the DNA damage in the tested groups. The genotoxic effect of the smoking habit, as well as its modifying effect on genome damage inflicted by other agents, have been firmly established. However, no statistically significant effect of smoking was found in our study, neither in the control nor in the hazard group. This lack of effect was seen in all DNA damage determinations, both direct (DNA strand breakage and alkali-labile lesions) and enzyme-combined (base damage) and did not depend on the comet parameters, which were taken as damage indicators.

DNA↗
PubMed2000

Determining the ages of comets from the fraction of crystalline dust.

The timescale for the accretion of bodies in the disk surrounding a young star depends upon a number of assumptions, but there are few observational constraints. In our own Solar System, measurements of meteoritic components can provide information about the inner regions of the nebula, but not the outer parts. Observations of the evolution of more massive protostellar systems (Herbig Ae/Be stars) imply that significant changes occur in the physical properties of their dust with time. The simplest explanation is that thermal annealing of the original, amorphous grains in the hot inner nebula slowly increases the fractional abundance of crystalline material over time. Crystalline dust is then transported outward, where it is incorporated into comets that serve as a long-term reservoir for dust disks, such as that surrounding Beta Pictoris. Here we show that when applied to our own Solar System, this process can explain observed variations in both the volatile and dusty components of comets, while also providing a natural indicator of a comet's mean formation age. Studies of comets with different dust contents can therefore be used to investigate the timescales of the early Solar System.

Carbon Monoxide↗
PubMed1996

Detection of acetylene in the infrared spectrum of comet Hyakutake.

Comets are rich in volatile materials, of which roughly 80% (by number) are water molecules. Considerable progress is being made in identifying the other volatile species, the abundances of which should enable us to determine whether comets formed primarily from ice-covered interstellar grains, or from material that was chemically processed in the early solar nebula. Here we report the detection of acetylene (C2H2) in the infrared spectrum of comet C/1996 B2 (Hyakutake). The estimated abundance is 0.3-0.9%, relative to water, which is comparable to the predicted solid-phase abundance in cold interstellar clouds. This suggests that the volatiles in comet Hyakotake may have come from ice-covered interstellar grains, rather than material processed in the accretion disk out of which the Solar System formed.

Acetylene↗
PubMed2005

A large dust/ice ratio in the nucleus of comet 9P/Tempel 1.

Comets spend most of their life in a low-temperature environment far from the Sun. They are therefore relatively unprocessed and maintain information about the formation conditions of the planetary system, but the structure and composition of their nuclei are poorly understood. Although in situ and remote measurements have derived the global properties of some cometary nuclei, little is known about their interiors. The Deep Impact mission shot a projectile into comet 9P/Tempel 1 in order to investigate its interior. Here we report the water vapour content (1.5 10(32) water molecules or 4.5 10(6) kg) and the cross-section of the dust (330 km2 assuming an albedo of 0.1) created by the impact. The corresponding dust/ice mass ratio is probably larger than one, suggesting that comets are 'icy dirtballs' rather than 'dirty snowballs' as commonly believed. High dust velocities (between 110 m s(-1) and 300 m s(-1)) imply acceleration in the comet's coma, probably by water molecules sublimated by solar radiation. We did not find evidence of enhanced activity of 9P/Tempel 1 in the days after the impact, suggesting that in general impacts of meteoroids are not the cause of cometary outbursts.

Journal Article↗