Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay

<p>Abstract</p> <p>Background</p> <p>Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell <it>Escherichia coli </it>by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide.</p> <p>Results</p> <p>Exposing the <it>E. coli </it>strain TG1 to CIP starting at a minimum inhibitory concentration (MIC) of 0.012 μg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 μg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 μg/ml but scarce after 10 μg/ml. The repair activity was not strictly related to viability. Four <it>E. coli </it>strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose.</p> <p>Conclusion</p> <p>This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.</p

Male Oxidative Stress Infertility (MOSI): Proposed Terminology and Clinical Practice Guidelines for Management of Idiopathic Male Infertility

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm’s potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause

Differential resistance of mammalian sperm chromatin to oxidative stress as assessed by a two-tailed comet assay

Protamines of eutherian species are cysteine-rich molecules that become cross-linked by disulfide bonds during epididymal transit, whereas the protamines of most marsupial species lack cysteine residuals. The present study made use of the differences in protamine structure between eutherian and metatherian mammal spermatozoa to examine the comparative resistance of sperm DNA to oxidative damage in three eutherian species (Mus musculus, Homo sapiens, Sus domesticus) and three metatherian species (Vombatus ursinus, Phascolarctos cinereus, Macropus giganteus). Sperm DNA fragmentation of samples exposed to increasing concentrations of hydrogen peroxide was assessed by means of the two-tailed comet assay. The sperm DNA of the marsupial species studied were significantly more sensitive to oxidative stress than the spermatozoa of eutherian species. Such susceptibility is consistent with the lack of disulfide cross-linking in marsupial sperm chromatin and suggests that the oxidation of thiols to disulfides for chromatin condensation during epididymal transit in eutherian mammals is likely to be important in order to provide stability and protect these cells from the genotoxic effects of adverse environments

Sperm Chromatin Dispersion (SCD) Assay

The sperm chromatin dispersion (SCD) test is a simple and rapid assay that detects sperm DNA fragmentation; it can be performed without the need of complex laboratory equipment. Spermatozoa enclosed in a thin inert matrix are incubated with an acid solution that denatures the DNA followed by an adapted lysing solution that removes the majority of nuclear proteins. This procedure results in DNA loops spreading out into the inert matrix, producing halos of chromatin. In comparison, human sperm with fragmented DNA do not produce halos or produce halos that are very small. This procedure has been validated in situ since only those spermatozoa without or with small halos are tagged by other sequential DNA breakage labeling assays. The availability of the SCD test as a commercialized kit (Halosperm®) provides clinicians with a user-friendly tool for routine use; the assay has now been employed in different clinical applications on a global basis for more than a decade. The utility of the SCD test is further enhanced when it is conducted dynamically in analyzing sperm DNA longevity; when spermatozoa are incubated in vitro, it is possible to reveal underlying DNA damage that is not apparent on initial assessment. The SCD test, thus, is a powerful and versatile methodology that not only facilitates easy assessment of sperm DNA quality in the clinic but also can be used in both basic and applied research related to human sperm DNA damage and organization

Dry biobanking as a conservation tool in the Anthropocene

Species are going extinct at an alarming rate, termed by some as the sixth mass extinction event in the history of Earth. Many are the causes for this but in the end, all converge to one entity - humans. Since we are the cause, we also hold the key to making the change. Any change, however, will take time, and for some species this could be too long. While working on possible solutions, we also have the responsibility to buy time for those species on the verge of extinction. Genome resource banks, in the form of cryobanks, where samples are maintained under liquid nitrogen, are already in existence but they come with a host of drawbacks. Biomimicry - innovation inspired by Nature, has been a huge source for ideas. Searching methods that Nature utilizes to preserve biological systems for extended periods of time, we realize that drying rather than freezing is the method of choice. We thus argue here in favor of preserving at least part of the samples from critically endangered species in dry biobanks, a much safer, cost-effective, biobanking approach. (C) 2020 Elsevier Inc. All rights reserved

DNA fragmentation of equine cumulus cells from Cumulus-Oocyte complexes submitted to vitrification and its relationship to the developmental competence of the oocyte

The objectives of this study were to evaluate the effect of vitrification on the DNA fragmentation rate of equine cumulus cells and to assess its relationship to oocyte in vitro maturation (IVM) after vitrification. Cumulus cells (CC) from 14 mares were recovered from COCs, previously submitted to vitrification (VIT) and IVM. The DNA fragmentation rate of the cumulus cells (CC-DF) was assessed using a chromatin dis-persion test. CC-DF rates between vitrified and control COCs were statistically com-pared by Student’s t- test. The rates of CC-DF from control COCs were lower than in vitrified COCs. The percentage of CC-DF was not significantly different (p> .05) be-tween groups of COCs able to reach metaphase II (MII > 0) and those in which oocyte maturation was not achieved (MII = 0). In conclusion, vitrification has a deleterious effect on the DNA fragmentation of equine cumulus cells; however, this parameter cannot be used as a predictor for IVM success after COCs vitrification

Investigation of pathology associated with Chlamydia pecorum infection in the male reproductive tract, and the effect on spermatogenesis and semen quality in the koala (Phascolarctos cinereus)

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morphology, suggestive of chronic exposure to C. pecorum infection and/or inflammation in the testis and epididymis, with residual pathology and lesions disrupting spermatogenesis and maturation of spermatozoa. This study specifically aimed to determine whether pathology associated with chlamydial infection in different regions of the male koala reproductive tract had an adverse effect on classical seminal parameters, sperm DNA quality and endocrine function (testosterone secretion) of naturally infected males. Semen from 58 sexually mature male koalas deemed not suitable for rehabilitation or treatment was assessed, in addition to undertaking a GnRH challenge to determine the androgenic capacity of the testis. Following euthanasia, tissue samples from testes, epididymis and prostate were evaluated for histopathology and real time polymerase chain reaction (qPCR). A significant difference in sperm concentration was observed between males with unilateral and bilateral testicular atrophy and C. pecorum infection (P = 0.011); and between males with unilateral atrophy and C. pecorum infection in one testis and bilateral normal testes with no C. pecorum infection (P = 0.008). No significant association was found for any other semen parameters when categorised by histopathology and C. pecorum tissue presence within the testes, epididymis and prostate. Plasma testosterone concentrations did not significantly differ between testicular histopathology diagnosis and/or C. pecorum infection status. This study suggests Chlamydia infection and inflammation may not be the predominant reason of disruption to spermatogenesis in the wild koala but rather testicular degeneration and atrophy, irrespective of Chlamydia infection, appears to be the primary reason of decreased sperm concentration.</p

Reduced sperm DNA longevity is associated with an increased incidence of still born; evidence from a multi-ovulating sequential artificial insemination animal model

Using a rabbit model, we assessed the influence of sperm DNA longevity on female reproductive outcomes