Control of the cell cycle during meiotic maturation of mouse oocytes

The overall aim of the work presented in this thesis is to better understand the molecular mechanisms underlying the progression of the meiotic division in mammalian oocytes. The first goal of this project was to investigate the relative contribution of securin and cyclin B1 to the control of the protease separase during the indeterminate period of metaphase II arrest. I found here that although there are conditions in which either securin or MPF can prevent chromosome disjunction, such as during meiosis I, securin is the predominant inhibitor of separase during metaphase II arrest. Thus, CDK1 pharmacological inhibition as well as antibody inhibition of CDK1/cyclin B1 binding to separase, both failed to induce sister chromatid disjunction. Instead, securin morpholino knockdown induced sister chromatid separation, which could be rescued by injection of securin cRNA. I also examined the effect of CDK1 and MAPK activities on securin stability. Inhibition of both kinases, but not either one alone, using roscovitine and UO126, induced the premature destruction of securin during prometaphase of meiosis I. However, this effect is not dependent on securin phosphorylation, given that mutagenesis of CDK1 and MAPK phosphorylation sites on securin did not affect its stability. Finally, I found that CDK1 and MAPK inhibition also causes the premature degradation of other APC/C substrates, such as cyclin B1 and geminin. Interestingly, only the D-box containing substrates and not Ken-box substrates were subject to such effect. However, this prometaphase I instability of the D-box substrates was apparently not mediated by APC/CCdc20 or APC/CCdh1, given that Cdc20 was also targeted for destruction and morpholino-knockdown of both Cdc20 and Cdh1 failed to rescue securin stability. Also, the inhibition of SCFβ-TrCP using the dominant negative mutant of its F-box protein (β-TrCPΔ) could not restore securin levels, suggesting it is not SCFβ-TrCP-dependent. Future work will explore whether this effect could be attributed to activation of some meiosis-specific APC/C co-activators or the core APC/C, which was recently reported of being able to directly interact with the D-box containing substrates independently of Cdc20 and Cdh1 (Passmore et al., 2003; Yamano et al., 2004)

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Cyclin A2 modulates kinetochore-microtubule attachment in meiosis II

Cyclin A2 is a crucial mitotic Cdk regulatory partner that coordinates entry into mitosis and is then destroyed in prometaphase within minutes of nuclear envelope breakdown. The role of cyclin A2 in female meiosis and its dynamics during the transition from meiosis I (MI) to meiosis II (MII) remain unclear. We found that cyclin A2 decreases in prometaphase I but recovers after the first meiotic division and persists, uniquely for metaphase, in MII-arrested oocytes. Conditional deletion of cyclin A2 from mouse oocytes has no discernible effect on MI but leads to disrupted MII spindles and increased merotelic attachments. On stimulation of exit from MII, there is a dramatic increase in lagging chromosomes and an inhibition of cytokinesis. These defects are associated with an increase in microtubule stability in MII spindles, suggesting that cyclin A2 mediates the fidelity of MII by maintaining microtubule dynamics during the rapid formation of the MII spindle