In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.

Late recurrences reduced the long-term efficacy of AF ablation, particularly in patients with PersAF and underlying cardiovascular diseases.

Background-The efficacy of radiofrequency ablation for atrial fibrillation (AF) in patients with left ventricular (LV) systolic dysfunction and isolated diastolic dysfunction is uncertain. Methods and Results-A prospective cohort of patients with normal and abnormal LV function underwent ablation for antiarrhythmic drug (AAD)-refractory AF. Three groups were compared: 111 patients with systolic dysfunction, defined as LV ejection fraction (LVEF) Յ40%; 157 patients with isolated diastolic dysfunction but preserved LVEF Ն50%; and 100 patients with normal LV function. The primary end point was AAD-free AF elimination at 1 year after ablation. This end point was achieved in 62% of patients with systolic dysfunction, 75% of those with diastolic dysfunction, and 84% of controls (Pϭ0.007). AF control on or off AADs was achieved in 76% of patients with systolic dysfunction, 85% of those with diastolic dysfunction, and 89% of controls (Pϭ0.08).In the systolic dysfunction group, 49% experienced an increase in LVEF by Ն5% after ablation, of which 64% achieved normal LVEF. In the diastolic dysfunction group, 30% of patients demonstrated at least 1 grade improvement in diastolic dysfunction. Multivariable analysis demonstrated an increased relative risk of arrhythmia recurrence of 1.8 (95% CI, 1.1 to 3.1; Pϭ0.02) in systolic dysfunction and 1.7 (1.0 to 2.7; Pϭ0.04) in isolated diastolic dysfunction compared with normal function. Conclusions-Although an ablative approach for AF in patients with systolic or diastolic dysfunction is associated with an increased long-term recurrence risk, there is potential for substantial quality-of-life improvement and LV functional benefit. (Circ Arrhythm Electrophysiol. 2011;4:724-732.)

AF ablation produces sustained QoL improvement at 2 years in patients with and without recurrence. AF-specific symptom assessment more accurately reflects ablative efficacy.

BackgroundPotential cardiovascular (CV) risks of testosterone replacement therapy (TRT) are currently a topic of intense interest. However, no studies have addressed CV risk as a function of the route of administration of TRT.MethodsTwo meta-analyses were conducted, one of CV adverse events (AEs) in 35 randomized controlled trials (RCTs) of TRT lasting 12 weeks or more, and one of 32 studies reporting the effect of TRT on serum testosterone and dihydrotestosterone (DHT).ResultsCV risks of TRT: Of 2,313 studies identified, 35 were eligible and included 3,703 mostly older men who experienced 218 CV-related AEs. No significant risk for CV AEs was present when all TRT administration routes were grouped (relative risk (RR) = 1.28, 95% confidence interval (CI): 0.76 to 2.13, P = 0.34). When analyzed separately, oral TRT produced significant CV risk (RR = 2.20, 95% CI: 1.45 to 3.55, P = 0.015), while neither intramuscular (RR = 0.66, 95% CI: 0.28 to 1.56, P = 0.32) nor transdermal (gel or patch) TRT (RR = 1.27, 95% CI: 0.62 to 2.62, P = 0.48) significantly altered CV risk. Serum testosterone/DHT following TRT: Of 419 studies identified, 32 were eligible which included 1,152 men receiving TRT. No significant difference in the elevation of serum testosterone was present between intramuscular or transdermal TRT. However, transdermal TRT elevated serum DHT (5.46-fold, 95% CI: 4.51 to 6.60) to a greater magnitude than intramuscular TRT (2.20-fold, 95% CI: 1.74 to 2.77).ConclusionsOral TRT produces significant CV risk. While no significant effects on CV risk were observed with either injected or transdermal TRT, the point estimates suggest that further research is needed to establish whether administration by these routes is protective or detrimental, respectively. Differences in the degree to which serum DHT is elevated may underlie the varying CV risk by TRT administration route, as elevated serum dihydrotestosterone has been shown to be associated with CV risk in observational studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-014-0211-5) contains supplementary material, which is available to authorized users.