Anesthetic and analgesic effects of an opioid-free, injectable protocol in cats undergoing ovariohysterectomy: A prospective, blinded, randomized clinical trial.

This study evaluated the effects of ketamine-dexmedetomidine-midazolam as part of an opioid-free, multimodal protocol in cats undergoing ovariohysterectomy. In a prospective, blinded, randomized clinical trial, cats received either 1 of 2 doses of ketamine [5 mg/kg body weight (BW), n = 10, K5 or 7 mg/kg BW, n = 13, K7] with midazolam (0.25 mg/kg BW) and dexmedetomidine (40 µg/kg BW) intramuscularly, intraperitoneal bupivacaine (2 mg/kg BW) and subcutaneous meloxicam (0.2 mg/kg BW) after surgery. Buprenorphine (0.02 mg/kg BW, intravenously) was administered if pain scores exceeded intervention scores with 2 pain scoring systems. Similar prevalence of rescue analgesia was observed (K5 = 6/10; K7 = 7/13) with significantly lower requirements in kittens (2/8) than adults (11/15). Tachypnea (K5 = 7/10 and K7 = 9/13) and desaturation (K5 = 3/10 and K7 = 4/13) were the 2 most common complications. Age influenced the prevalence of rescue analgesia. Most adult cats required opioids for postoperative pain relief.

Effets anesthésiants et analgésiques d'un protocole injectable sans opioïde chez des chats soumis à une ovario-hystérectomie : essai clinique prospectif, randomisé, à l'aveugle. Lors de la présente étude nous avons évalué les effets de la combinaison kétamine-dexmedetomidine-midazolam comme élément d'un protocole multimodal sans opioïde chez des chats soumis à une ovario-hystérectomie. Dans un essai clinique prospectif, randomisé, à l'aveugle, des chats reçurent une des deux doses de kétamine [5 mg/kg poids corporel (BW), n = 10, K5 ou 7 mg/kg BW, n = 13, K7] avec du midazolam (0,25 mg/kg BW) et du dexmedetomidine (40 µg/kg BW) par voie intramusculaire, de la bupivacaine par voie intrapéritonéale (2 mg/kg BW) et du meloxicam sous-cutané (0,2 mg/kg BW) après la chirurgie. De la buprenorphine (0,02 mg/kg BW, par voie intraveineuse) fut administrée si les pointages de douleur excédaient les pointages d'intervention avec deux systèmes de pointage de la douleur. Une prévalence similaire d'analgésie de secours fut observée (K5 = 6/10; K7 = 7/13) avec des demandes significativement moindres chez les chatons (2/8) que chez les adultes (11/15). De la tachypnée (K5 = 7/10 et K7 = 9/13) et de la désaturation (K5 = 3/10 et K7 = 4/13) étaient les deux complications les plus fréquentes. L'âge influençait la prévalence de l'analgésie de secours. La plupart des chats adultes ont requis des opioïdes pour soulager la douleur post-opératoire.(Traduit par Dr Serge Messier).

Detection of oriC-Independent Replication in Escherichia coli Cells.

In bacteria, replication of the chromosome is normally initiated following the binding of DnaA proteins to the oriC region. However, under certain circumstances, replication can also be initiated independent of the oriC/DnaA system. This is the case, for example, in Escherichia coli cells lacking RNase HI (rnha mutants) or type 1A topoisomerase activity (topA topB mutants). Here, we present a protocol in which replication from the oriC/DnaA system is first inhibited by the addition of the protein synthesis inhibitor, spectinomycin, to exponentially growing bacterial cell cultures. The thymidine analog, 5-ethynyl-2'-deoxyurdine (EdU) is then added to the cells, and after 1 h the cells are fixed and the Alexa Fluor® 488 dye is conjugated to EdU by the click-iT® reaction. The oriC-independent replication is detected in fixed cells by flow cytometry and can be visualized by fluorescence microscopy.

Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low temperatures that stabilizes hyper-negatively supercoiled DNA.

Constitutive stable DNA replication in Escherichia coli cells lacking type 1A topoisomerase activity.

Type 1A topoisomerases (topos) are ubiquitous enzymes involved in supercoiling regulation and in the maintenance of genome stability. Escherichia coli possesses two type 1A enzymes, topo I (topA) and topo III (topB). Cells lacking both enzymes form very long filaments and have severe chromosome segregation and growth defects. We previously found that RNase HI overproduction or a dnaT::aph mutation could significantly correct these phenotypes. This leads us to hypothesize that they were related to unregulated replication originating from R-loops, i.e. constitutive stable DNA replication (cSDR). cSDR, first observed in rnhA (RNase HI) mutants, is characterized by its persistence for several hours following protein synthesis inhibition and by its requirement for primosome components, including DnaT. Here, to visualize and measure cSDR, the incorporation of the nucleotide analog ethynyl deoxyuridine (EdU) during replication in E. coli cells pre-treated with protein synthesis inhibitors, was revealed by "click" labeling with Alexa Fluor(®) 488 in fixed cells, and flow cytometry analysis. cSDR was detected in rnhA mutants, but not in wild-type strains, and the number of cells undergoing cSDR was significantly reduced by the introduction of the dnaT::aph mutation. cSDR was also found in topA, double topA topB but not in topB null cells. This result is consistent with the established function of topo I in the inhibition of R-loop formation. Moreover, our finding that topB rnhA mutants are perfectly viable demonstrates that topo III is not uniquely required during cSDR. Thus, either topo I or III can provide the type 1A topo activity that is specifically required during cSDR to allow chromosome segregation.