Antioxidant molecules as a source of mitigation of antibiotic resistance genes dissemination

Escherichia coli is the most commonly identified human pathogen, and a prominent microorganism of the gut microbiota. Acquired resistance to antibiotics in that species is mainly driven by horizontal gene transfer, and mainly by plasmid acquisition. The main concern nowadays corresponds to the acquisition of extended-spectrum ß-lactamases of the CTX-M-type in E. coli, a worldwide observed phenomenon. Plasmids encoding CTX-M enzymes are of different scaffolds, and conjugate at different frequencies. Here we showed that the conjugation rates of several plasmid types encoding broad-spectrum ß-lactamases are increased when the E. coli donor strain is exposed to sub-inhibitory concentrations of diverse orally-given antibiotics, including fluoroquinolones such as ciprofloxacin and levofloxacin, but also trimethoprim, and nitrofurantoin. This study provided insights into underlying mechanisms leading to increase plasmid conjugation frequency in relation with DNA synthesis inhibitors-type antibiotics, involving reactive oxygen species (ROS) production and probably increased expression of genes involved in the SOS response. Furthermore, we showed that some antioxidant molecules currently approved for unrelated clinical uses such as edaravone, p-Coumaric acid and N-acetylcysteine may antagonize the inducibility effect of antibiotics in term of increased plasmid conjugation rates. These results suggest that several antioxidative molecules might be used in combination with those “inducer” antibiotics to mitigate the unwanted increased resistance plasmid dissemination.

Synchronization of human retinal pigment ephitilial-1 (RPE-1) cells in mitosis

ABSTRACTHuman retinal pigment ephitilial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4/6 inhibitor PD 0332991 (palbociclib) and the microtubule depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Furthermore, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.

Ranking of Major Classes of Antibiotics for Activity against Stationary Phase Pseudomonas aeruginosa and Identification of Clinafloxacin + Cefuroxime + Gentamicin Drug Combination that Eradicates Persistent P. aeruginosa Infection in a Murine Cystic Fibrosis Model

Pseudomonas aeruginosa can cause serious persistent infections such as ventilator-associated pneumonia, sepsis, biofilm-related infections as in cystic fibrosis (CF) patients. Although CF lung infections can be treated with antibiotics, full clearance is difficult due to P. aeruginosa persistence. While antibiotic activity against growing P. aeruginosa is well documented, their activity against the non-growing persisters enriched in stationary phase cultures has not been well studied. Here, we systematically evaluated and ranked the six major classes of antibiotics, cell wall and cell membrane inhibitors, protein synthesis inhibitors, DNA synthesis inhibitors, RNA synthesis inhibitors, sulfa drugs, and nitrofurantoin, for their activity against both growing and persister forms of P. aeruginosa using colony forming count (CFU) and SYBR Green I/Propidium Iodide (PI) viability assay. Among the six major classes of antibiotics, cell wall and cell membrane inhibitors (Cefuroxime and Colistin), DNA synthesis inhibitors (Clinafloxacin) and sulfa drugs (Sulfamethoxazole) had good activity against stationary phase cells. In contrast, protein synthesis inhibitors (Gentamicin), RNA synthesis inhibitor (Rifampicin) and Nitrofurantoin had relatively poor activity against the stationary phase P. aeruginosa but relatively high activity against log phase P. aeruginosa. Clinafloxacin is the only single drug that could completely kill all (109 CFU) stationary phase cells in a 4 day drug exposure. The Cefuroxime + Gentamicin+ Clinafloxacin combination could kill all biofilm bacteria in 2 days whereas the clinically used drug combination Cefuroxime + Gentamicin + Colistin only partially killed the biofilm bacteria with 103 CFU remaining. In a murine persistent CF lung infection model, only Cefuroxime + Gentamicin+ Clinafloxacin cleared all bacteria in the infected lungs, whereas Clinafloxacin alone, or Cefuroxime + Clinafloxacin, or the current recommended drug combination Cefuroxime + Gentamicin, all failed to completely clear the bacterial load in the lungs. The complete sterilization of the bacterial load is a property of Clinafloxacin combination, as Cefuroxime + Gentamicin+ Levofloxacin combination was unable to clear the bacterial load in the lungs. Our findings demonstrate the importance of persister drug clinafloxacin, offer new therapeutic approaches for more effective treatment of persistent P. aeruginosa infections, and may have implications for treating other persistent infections.

Principles of chemotherapy

Systemic cancer treatment stems initially from empirically discovered DNA synthesis inhibitors, which either deplete the cell of nucleotides, induce cross-link, or cause DNA single and double strand breaks or impair the cellular machinery of DNA repair, using mechanistically diverse drugs. A period of enlightenment followed, with anticancer drug development driven by an increased understanding of enzymes and pathways involved in cell signalling, control of angiogenesis, and epigenetics. This provided a parallel path towards precision cancer medicine where specific drugs can be targeted to patients with particular mutations. These include point mutations in RAS, which are used to exclude colorectal cancer patients from being treated with epidermal growth factor inhibitors; chromosomal translocations encoding fusion proteins which are cancer specific and serve as novel drug targets (e.g. BCR/ABL and imatinib, or EML4-ALK fusion oncogene and crizotinib). More recently, there has been a reanimation of immune approaches to cancer therapy with the clinical introduction of immune checkpoint inhibitors, designer T cells, and patient-specific antitumour vaccines. What next? It may be that next-generation sequencing provides an endless stream of so-called actionable mutations that permits tailored application of mutation-specific drugs, but so far there is little evidence of clinical benefit from such therapies.

Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy.

Effect of N-hydroxyurea, mitomycin C and actinomycin D on tumour formation on the leaves of Kalanchoe daigremontiana

The leaves of <em>Kalanchoe daigremontiana</em> wounded and infected with <em>Agrobacterium tumefaciens</em> were treated with single doses of inhibitors (hydroxyurea - 190, mitomycin - 0.5, actinomycin - 2 µ,g per leaf). After delaying the time' of dosage of inhibitors during five days after inoculation, changes in susceptibility of the system to antitumorous activity of analysed compounds were observed. In several hours after inoculation (period of the bacteria metabolic activity in wounds) all the inhibitors prevent strongly the tumour formation. At the time between 14 and 72 hours after inoculation, including the phase of tumour induction, the system becomes sensitive to the DNA synthesis inhibitors, particularly hydroxyurea. The intensified action of actinomycin appears again only about 60 hours after inoculation and lasts till the end of experiment (the initiation of the transformed plant cell proliferation). According to the literature the antitumorous effect of inhibitors could be connected with their action on the bacteria metabolism inside the host tissue. The activities of hydroxyurea and mitomycin in the second period correspond with the intensive DNA synthesis in plant cells, which is induced by wounding. The effect of actinomycin D in 60 hours after inoculation could depend upon the inhibition of the proliferation of the transformed host cells.