Modelling the comet assay

The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters.

Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success

Objective: To evaluate sperm DNA fragmentation and semen parameters to diagnose male factor infertility and predict pregnancy after IVF.
Design: Prospective study.
Setting: Academic research laboratory.
Patient(s): Seventy-five couples undergoing IVF and 28 fertile donors.
Intervention(s): Sperm DNA fragmentation was measured by the alkaline Comet assay in semen and sperm after density gradient centrifugation (DGC). Binary logistic regression was used to analyze odds ratios (OR) and relative risks (RR) for IVF outcomes.
Main Outcome Measure(s): Semen parameters and sperm DNA fragmentation in semen and DGC sperm compared with fertilization rates, embryo quality, and pregnancy.
Result(s): Men with sperm DNA fragmentation at more than a diagnostic threshold of 25% had a high risk of infertility (OR: 117.33, 95% confidence interval [CI]: 12.72–2,731.84, RR: 8.75). Fertilization rates and embryo quality decreased as sperm DNA fragmentation increased in semen and DGC sperm. The risk of failure to achieve a pregnancy increased when sperm DNA fragmentation exceeded a prognostic threshold value of 52% for semen (OR: 76.00, CI: 8.69–1,714.44, RR: 4.75) and 42% for DGC sperm (OR: 24.18, CI: 2.89–522.34, RR: 2.16).
Conclusion(s): Sperm DNA testing by the alkaline Comet assay is useful for both diagnosis of male factor infertility and prediction of IVF outcome.

Divergence of chemical function in the alkaline phosphatase superfamily: Structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and P-32-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

Kinetics and mechanism of a macrocyclic chromium(III) complex oxidation to chromium(IV) by hexacyanoferrate(III) in strongly alkaline media

Oxidation of the macrocyclic Cr(III) complex cis-[Cr(cycb)(OH)(2)](+), where cycb = rac-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane, by an excess of the hexacyanoferrate( III) in basic solution, slowly produces Cr(V) species. These species, detected using e.p.r. spectroscopy, are stable under ambient conditions for many hours, and the hyperfine structure of the e.p.r. spectrum is consistent with the interaction of the d-electron with four equivalent nitrogen nuclei. Electro-spray ionization mass spectrometry suggests a concomitant oxidation of the macrocyclic ligand, in which double bonds and double bonded oxygen atoms have been introduced. By comparison basic chromate(III) solutions are oxidized rapidly to chromate(VI) by hexacyanoferrate(III) without any detectable generation of stable Cr(V) intermediates.

Alkaline hydrolysis of trinitrotoluene, TNT

The kinetics of the alkaline hydrolysis of trinitrotoluene, TNT, in an aqueous solution is a possible approach to destroying the active agent in unwanted munitions. The kinetics are shown to have a rapid initial step, step A, in which a highly coloured species, X (lambda(max) = 450 nm) is formed via an equilibrium reaction: TNT + OH- double left right arrow X. The bimolecular rate constant for the forward part of this equilibrium process, k(1), is: 0.099 +/- 0.004, 0.32 +/- 0.02 and 1.27 +/- 0.05 dm(3) mol(-1) s(-1), at 25, 40 and 60degreesC, respectively. The activation energy for the forward process is 60 kJ mol(-1). The first-order rate constant for the reverse of this process, k(-1), is: (5.3 +/- 2.6) x 10(-4), (1.2 +/- 1.0) x 10(-3) and (7.7 +/- 2.9) x 10(-3) s(-1) at 25, 40 and 60degreesC, respectively. The activation energy for the overall equilibrium process (k(1)/k(-1)) is ca. -5 kJ mol(-1). The subsequent alkaline hydrolysis of X to form the final product P, i.e. step B, is much slower than step A and appears to comprise two processes coupled in series, i.e. steps B1 (X +2OH(-) --> Z) and B2 (Z+OH- --> P). At 25degreesC, Step B1 appears rate determining throughout the decay process. At 45 degreesC and, more so, at 60degreesC, step B appears increasingly biphasic with increasing alkaline concentrations, as step B2 begins to compete with step B1 for position as the rate determining step. The trimolecular rate constant for step B1 is: 0.017 +/- 0.001, 0.0085 +/- 0.0002 and 0.0011 +/- 0.0001 dm(6) mol(-2) s(-1) at 25, 40 and 60degreesC, respectively, and the process has an activation energy of 64 kJ mol(-1). The transition from uniform kinetics, described by step B1, to mixed kinetics, described by steps B1 and B2, as the reaction temperature and alkali concentration are increased most likely occurs because (a) step B2 has a lower activation energy than B1, although it was not possible to measure the former parameter, and (b) step B2 has a lower (1st) order dependence upon [OH-] compared with that of step B1 (2nd). The bimolecular rate constant for step B2 is 0.0035 +/- 0.03 dm(3) mol(-1) s(-1) at 60degreesC. A brief NMR study of the initial hydrolysis product in water, acetone and chloroform, coupled with UV/visible spectra, provides evidence that species X is a Meisenheimer complex.

Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in Escherichia coli

Background: In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis.
Results: Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress.
Conclusions: Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli.

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78ngmL and the CCß to be 1ngmL. Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC-MS/MS showed a high correlation (R=0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1ngmL, and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1ngmL. This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1µgL. This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.

Electrooxidation of methanol in an alkaline fuel cell: determination of the nature of the initial adsorbate

It is essential to correctly determine the nature of the initial adsorbate in order to calculate the pathway for any given reaction. Recent literature provides conflicting information on the first step in the methanol decomposition pathway. This work sets out to establish what role the solution and the surface have to play in the initial adsorption-deprotonation process. Density functional theory (DFT) calculations, in combination with a cluster-continuum model approach are used to resolve the nature of the adsorbing species. We show that methanol is the dominant species in solution over methoxide, and also has a smaller barrier to adsorption. The nature of the surface species is revealed to be a methanol-OH complex.

The Role of Water and Adsorbed Hydroxyls on Ethanol Electrochemistry on Pd: New Mechanism, Active Centers and Energetics for Direct Ethanol Fuel Cell Running in Alkaline Medium

First principles calculations with molecular dynamics are
utilized to simulate a simplified electrical double layer formed in the
active electric potential region during the electrocatalytic oxidation of
ethanol on Pd electrodes running in an alkaline electrolyte. Our
simulations provide an atomic level insight into how ethanol oxidation
occurs in fuel cells: New mechanisms in the presence of the simplified
electrical double layer are found to be different from the traditional
ones; through concerted-like dehydrogenation paths, both acetaldehyde
and acetate are produced in such a way as to avoid a variety of
intermediates, which is consistent with the experimental data obtained
from in situ FTIR spectroscopy. Our work shows that adsorbed OH on
the Pd electrode rather than Pd atoms is the active center for the
reactions; the dissociation of the C−H bond is facilitated by the
adsorption of an OH− anion on the surface, resulting in the formation
of water. Our calculations demonstrate that water dissociation rather than H desorption is the main channel through which
electrical current is generated on the Pd electrode. The effects of the inner Helmholtz layer and the outer Helmholtz layer are
decoupled, with only the inner Helmholtz layer being found to have a significant impact on the mechanistics of the reaction. Our
results provide atomic level insight into the significance of the simplified electrical double layer in electrocatalysis, which may be
of general importance.

Two potential clinical applications of the alkaline single-cell gel electrophoresis assay .1. Human bladder washings and transitional cell carcinoma of the bladder .2. Human sperm and male infertility

Part 1: The alkaline single-cell gel electrophoresis (comet) assay was used to analyse the integrity and DNA content of exfoliated cells extracted from bladder washing specimens from 9 transitional cell carcinoma patients and 15 control patients. DNA damage, as expressed by % tail DNA and tail moment values, was observed to occur in cells from both control and bladder cancer samples. The extent of the damage was, however, found to be significantly greater in the cancer group than in the control group. Comet optical density values were also recorded for each cell analysed in the comet assay and although differences observed between tumour grades were not found to be statistically significant, the mean comet optical density value was observed to be greater in the cancer group than in the control population studied, These preliminary results suggest that the comet assay may have potential as a diagnostic tool and as a prognostic indicator in transitional cell carcinoma, Part 2: Baseline DNA damage in sperm cells from 13 normozoospermic fertile males, 17 normozoospermic infertile males and 11 asthenozoospermic infertile males were compared using a modified alkaline comet assay technique. No significant difference in the level of baseline DNA damage was observed between the 3 categories of sperm studied; however the untreated sperm cells were observed to display approximately 20% tail DNA. This is notably higher than the background DNA damage observed in somatic cells where the % tail DNA is normally less than 5%. Sperm from the 3 groups of men studied were also compared for sensitivity to DNA breakage, using the modified alkaline comet assay, following X-ray irradiations (5, 10 and 30 Gy) and hydrogen peroxide treatments (40, 100 and 200 mu M). Significant levels of X-ray-induced damage were found relative to untreated control sperm in the two infertile groups following 30 Gy irradiation. Significant damage in hydrogen peroxide-treated sperm was observed in sperm from fertile samples, at 200 mu M and in infertile samples at 100- and 200-mu M doses relative to controls. These results therefore indicate that fertile sperm samples are more resistant to X-ray- and hydrogen peroxide-induced DNA breakage than infertile samples. Further studies involving greater numbers of individuals are currently in progress to confirm these findings.