Cytochrome c (Cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc₁ complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that Cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I Cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H₂O₂ were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψ_m), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψ_m hyperpolarization, ROS production, and apoptosis.

Cytochrome c (Cyt c ) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue‐specific phosphorylations of Cyt c in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cyt c purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cyt c purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cyt c at 1.55 Å and suggest that it spatially matches S47‐phosphorylated Cyt c , making it a good model system. Both S47‐phosphorylated and phosphomimetic Cyt c showed a lower oxygen consumption rate in reaction with isolated Cyt c oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47‐phosphorylated and phosphomimetic Cyt c showed lower caspase‐3 activity. Furthermore, phosphomimetic Cyt c had decreased cardiolipin peroxidase activity and is more stable in the presence of H ₂ O ₂ . Our data suggest that S47 phosphorylation of Cyt c is tissue protective and promotes cell survival in the brain.—Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.‐A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine‐47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase‐3 activity. FASEB J. 33, 13503‐13514 (2019). www.fasebj.org

Background: The population of women undergoing abdominal myomectomy for symptomatic large fibroid uterus is unique. We seek to characterize the timing, risk factors as well as the presenting symptoms which led patients to undergo repeat surgery in this patient population. Methods and findings: We followed 592 patients who underwent an abdominal myomectomy from March 1998 to June 2010 at St. Vincent’s Catholic Medical Center and presented later during the study period with a recurrence of symptoms attributable to a reemergence of fibroids and who chose to undergo repeat surgical management. Twelve percent of patients exhibited symptoms of fibroid uterus which led to reoperation within the study period. The mean age at repeat surgery was 44.1 ± 0.6 years old (n = 69) and the mean time between operations was 7.9 ± 0.3 years. Presentation was variable but included bleeding, pain and infertility. Patients presented for surgery with a significantly smaller sized uterus than at their initial surgery. Timing between surgeries correlated with age at initial surgery and uterine size but race, number of fibroids, aggregate weight of fibroids removed, operative time or blood loss at the initial surgery did not correlate. Data is suggestive that intraperitoneal triamcinolone may reduce reoperation rates but not timing of recurrence. Conclusion: These results may help in counseling patients, particularly younger women, on the risks of fibroid recurrence necessitating repeat surgery. Further research is necessary to assess if triamcinolone can alter fibroid reurrence in patients who undergo uterus sparing procedures.

To determine whether oral norethindrone acetate is superior to combined oral contraceptives (OCP) in delaying menstruation and preventing breakthrough bleeding when started late in the cycle. This article comprises of a case control study followed by a pilot randomized controlled study. In the first study, four women who presented late in their cycle and desired avoiding vaginal bleeding within 10 days before a wedding were started on norethindrone 5 mg three times daily and compared to age matched controls started on OCPs. Subsequently, a randomized controlled pilot study (n = 50) comparing OCPs to norethindrone for the retiming of menses was conducted. Percentage of women reporting spotting were compared with level of statistical significance set at p < 0.05. Of the norethindrone treated group, only 2 women (8%) reported spotting compared with 10 women (43%) in the control group (p < 0.01). Norethindrone recipients experienced significant weight gain, which resolved after cessation of therapy and had heavier withdrawal bleed (p < 0.04) when compared to controls. Patient satisfaction was significantly higher in the norethindrone group, with 80% willing to choose this method again. Time to conceive was significantly shorter in the norethindrone group (p < 0.03). Norethindrone, begun on or before cycle day 12, is superior for women who desire to avoid breakthrough bleeding and maintain fertility when compared to OCPs. It is an ideal approach in patients presenting late in their cycle and who desire delaying menses as well as in circumstances when even minute amounts of breakthrough bleeding cannot be tolerated. Clinicaltrials.gov NCT03594604 , July 2018. Retrospectively registered.