Structural Investigations of the Inhibition of Escherichia coli AmpC β-Lactamase by Diazabicyclooctanes

β-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli, but their use is increasingly compromised by β-lactamases, including the chromosomally encoded class C AmpC serine-β-lactamases (SBL). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimise the DBO core. We report kinetic and structural studies, including four high resolution crystal structures, concerning inhibition of the AmpC serine-β-lactamase from E. coli (AmpCEC) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometry-based assays were used to study their mechanisms of AmpCEC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (Kiapp 0.69 μM) against AmpCEC compared to the other DBOs (Kiapp 5.0-7.4 μM) due to an ∼ 10 fold accelerated carbamoylation-rate. However, zidebactam also has an accelerated off-rate and with sufficient preincubation time all the DBOs manifest similar potencies. Crystallographic analyses indicate a greater conformational freedom of the AmpCEC-zidebactam carbamoyl-complex compared to those for the other DBOs. The results suggest carbamoyl-complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.

The Effect of Preincubation Time and Myo-inositol Supplementation on the Quality of Mouse MII Oocytes

Background: It is demonstrated that optimal preincubation time improves oocyte quality, fertilization potential and developmental rate. This study aimed to evaluate the effect of preincubation time in the simple and myo-inositol supplemented medium on the oocyte quality regarding oxidative stress and mitochondrial alteration. Methods: Cumulus oocyte complexes (COCs) retrieved from superovulated NMRI mice were divided in groups of 0, 4 and 8 hr preincubation time in the simple and 20 mmol/L myo-inositol supplemented media. Intracellular reactive oxygen species (H2O2), glutathione (GSH), mitochondrial membrane potential (MMP), ATP content, and mitochondrial amount were measured and analyzed in experimental groups. One-way ANOVA and Kruskal-Wallis were respectively used for parametric and nonparametric variables. Statistical significance was defined as p<0.05. Results: In comparison to control group, variables including ROS, GSH, mitochondrial amount, fertilization and developmental rates were significantly changed after 4 hr of preincubation in the simple medium, while MMP decreased following 8 hr of preincubation in the simple medium (p˂0.001). Preincubation of oocytes up to 8 hr in the simple medium could not decrease ATP content. For both 4 and 8 hr preincubation times, myo-inositole could decrease H2O2 and increase GSH and MMP levels and consequently could improve fertilization rate compared to oocytes preincubated in the simple culture. Conclusion: It seems that 4 hr or more preincubation time can decrease the oocyte quality and lead to reduced oocyte fertilization and developmental potential. Howevere, myo-inositol may prevent oocyte quality reduction and improve fertilization potential in comparision to the equivalent simple groups.

Products of Sulfide/Selenite Interaction Possess Antioxidant Properties, Scavenge Superoxide-Derived Radicals, React with DNA, and Modulate Blood Pressure and Tension of Isolated Thoracic Aorta

Selenium (Se), an essential trace element, and hydrogen sulfide (H2S), an endogenously produced signalling molecule, affect many physiological and pathological processes. However, the biological effects of their mutual interaction have not yet been investigated. Herein, we have studied the biological and antioxidant effects of the products of the H2S (Na2S)/selenite (Na2SeO3) interaction. As detected by the UV-VIS and EPR spectroscopy, the product(s) of the H2S-Na2SeO3 and H2S-SeCl4 interaction scavenged superoxide-derived radicals and reduced ⋅cPTIO radical depending on the molar ratio and the preincubation time of the applied interaction mixture. The results confirmed that the transient species are formed rapidly during the interaction and exhibit a noteworthy biological activity. In contrast to H2S or selenite acting on their own, the H2S/selenite mixture cleaved DNA in a bell-shaped manner. Interestingly, selenite protected DNA from the cleavage induced by the products of H2S/H2O2 interaction. The relaxation effect of H2S on isolated thoracic aorta was eliminated when the H2S/selenite mixture was applied. The mixture inhibited the H2S biphasic effect on rat systolic and pulse blood pressure. The results point to the antioxidant properties of products of the H2S/selenite interaction and their effect to react with DNA and influence cardiovascular homeostasis. The effects of the products may contribute to explain some of the biological effects of H2S and/or selenite, and they may imply that a suitable H2S/selenite supplement might have a beneficial effect in pathological conditions arisen, e.g., from oxidative stress.

Evaluation of sperm motility with CASA-Mot: which factors may influence our measurements?

Computer-aided sperm analysis (CASA) is now routinely used in IVF clinics, animal breeding centres and research laboratories. Although CASA provides a more objective way to evaluate sperm parameters, a significant number of factors can affect these measurements. This paper classifies these factors into four categories: (1) sample and slide (e.g. preincubation time, type of specimen and type of chamber slide); (2) microscope (e.g. light source and microscope stage); (3) hardware and software, including the settings of each system; and (4) user-related factors. We review the effects of the different factors in each category on the measurements made and emphasise the need to take measures to standardise evaluations. The take-home message of the present article is that there are several commercial and useful CASA systems, and all are appropriate for routine analysis. Non-commercial systems may also be good choices when the user needs to adapt the device to specific experimental conditions. In both cases (commercial and non-commercial), it is important that standard protocols are put in place for evaluation, as well as methods to validate the system.

Effects of novel quinoid thiosulfonate derivative on ADP-induced platelet aggregation

Recently, in a large scale screening test, we have found that S-((1,4-dimethoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl)4-aminobenzenesulfonothioate, a chemically synthesized thiosulfonate derivative of quinone, possessed an antiplatelet activity. To elucidate the mechanism of its antiplatelet action, a series of experiments were performed. The compound was found to inhibit the ADP-induced platelet aggregation. The inhibitory effect was dose-dependent on concentration and preincubation time. The derivative also disaggregated the preformed platelet aggregates induced by ADP.

The effect of the time interval between injection and parthenogenetic activation on the spindle formation and the in vitro developmental potential of somatic cell nuclear-transferred rat oocytes

SummaryWe examined the optimal conditions for somatic cell nuclear transfer (SCNT) in the rat. First, we examined the effect of preincubation time before activation on SCNT rat oocytes produced in the presence of MG132 with regard to spindle formation and the potential to develop into blastocysts. The spindles of SCNT oocytes continued to elongate with an increase in the culture duration and, in approximately half of oocytes, the chromosomes were distributed along the spindles at 120 min after incubation. Such abnormal spindle formation in SCNT oocytes is a possible reason for the low developmental potential of SCNT rat oocytes. To inhibit the formation of abnormal spindle formation, we examined secondly the developmental potential of rat SCNT oocytes that had been preincubated with nocodazole and demecolcine instead of MG132. The developmental rates in SCNT oocytes, however, were decreased. For successful rat somatic cell cloning, two steps might be required: (1) to culture the somatic cell nuclei for a sufficient time in MII oocyte cytoplasm to enhance nuclear reprogramming; and (2) to induce normal spindle formation with normal chromosomal construction.