Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases.

Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O2, or 2) solvent remains bound to iron but changes its equilibrium position to allow O2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H2O versus D2O) on two stilbenoid-cleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (kH/kD ∼ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (kH/kD ∼ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5-8.5 and that the Fe(II)-aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism.

Preparation and characterization of metal-substituted carotenoid cleavage oxygenases.

Carotenoid cleavage oxygenases (CCO) are non-heme iron enzymes that catalyze oxidative cleavage of alkene bonds in carotenoid and stilbenoid substrates. Previously, we showed that the iron cofactor of CAO1, a resveratrol-cleaving member of this family, can be substituted with cobalt to yield a catalytically inert enzyme useful for trapping active site-bound stilbenoid substrates for structural characterization. Metal substitution may provide a general method for identifying the natural substrates for CCOs in addition to facilitating structural and biophysical characterization of CCO-carotenoid complexes under normal aerobic conditions. Here, we demonstrate the general applicability of cobalt substitution in a prototypical carotenoid cleaving CCO, apocarotenoid oxygenase (ACO) from Synechocystis. Among the non-native divalent metals investigated, cobalt was uniquely able to stably occupy the ACO metal binding site and inhibit catalysis. Analysis by X-ray crystallography and X-ray absorption spectroscopy demonstrate that the Co(II) forms of both ACO and CAO1 exhibit a close structural correspondence to the native Fe(II) enzyme forms. Hence, cobalt substitution is an effective strategy for generating catalytically inert but structurally intact forms of CCOs.

Standardizing the Dosage and Timing of Dexamethasone for Postoperative Nausea and Vomiting Prophylaxis at a Safety-Net Hospital System.

BACKGROUND: A single dose of dexamethasone is routinely given during general anesthesia for postoperative nausea and vomiting (PONV) prophylaxis, although the exact dosage and timing of administration may vary between practitioners. The authors aimed to standardize the dosage and timing of this medication when given to adult patients undergoing general anesthesia for elective surgery. METHODS: Baseline data for 7,483 preintervention cases were analyzed. The researchers attempted to use a standard dose of 8 to 10 mg induction of anesthesia, which, based on a literature review, was effective for PONV prophylaxis, had a similar safety profile as a 4 to 5 mg dose (including in diabetic patients), and may confer additional benefits such as improved prophylaxis and quality of recovery. The interventions included standardizing the medication concentration vials, altering electronic health record quick-select button options, simplifying the intraoperative charting process, and educating the anesthesia providers. The research team then tracked compliance with the standard of care for 2,167 cases after the interventions. RESULTS: Overall compliance with the standard of care increased from 21.2% preintervention to 53.7% postintervention. The number of patients not receiving dexamethasone was reduced from 29.7% to 19.4%. Patients receiving a compliant dose at a noncompliant time increased from 16.3% to 23.8%. Postanesthesia care unit antiemetic administration also decreased after the interventions. CONCLUSION: This study showed improvements in compliance with the dosage of medication with the interventions. However, compliance with the timing of administration remains challenging.

Understanding breast cancer disparities-a multi-scale challenge.

Despite convergence of overall breast cancer incidence rates between European American (EA) and African American (AA) women, disparities in mortality persist. The factors contributing to differences in mortality rates across population groups remain controversial and range from population genetics to sociodemographic influences. This review explores the complex multi-factorial nature of tumor-intrinsic and -extrinsic factors that impact the biology and clinical outcomes of breast cancer patients. In addition to summarizing the current state of breast cancer disparities research, we also motivate the development of integrative multi-scale approaches involving interdisciplinary teams to tackle this complex clinical challenge.