Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance

Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phos-phatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells

Constant regulation of both the MPF amplification loop and the Greatwall-PP2A pathway is required for metaphase II arrest and correct entry into the first embryonic cell cycle.

International audienceRecent results indicate that regulating the balance between cyclin-B-Cdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-B-Cdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist regarding the regulation of this balance during M phase and interphase. Here, we demonstrate that constant regulation of the cyclin-B-Cdc2 amplification loop is required for correct mitotic division and to promote correct timing of mitotic entry. Our results show that removal of Cdc25 from metaphase-II-arrested oocytes promotes mitotic exit, whereas depletion of either Myt1 or Wee1 in interphase egg extracts induces premature mitotic entry. We also provide evidence that, besides the cyclin-B-Cdc2 amplification loop, the Greatwall-PP2A pathway must also be tightly regulated to promote correct first embryonic cell division. When PP2A is prematurely inhibited in the absence of cyclin-B-Cdc2 activation, endogenous cyclin-A-Cdc2 activity induces irreversible aberrant mitosis in which there is, first, partial transient phosphorylation of mitotic substrates and, second, subsequent rapid and complete degradation of cyclin A and cyclin B, thus promoting premature and rapid exit from mitosis

The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A.

International audienceInitiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts

Greatwall maintains mitosis through regulation of PP2A

Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B–Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B–Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B–Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B–Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A-dependent dephosphorylation of cyclin B–Cdc2 substrates is increased in GW-depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B–Cdc2 but also by the regulation of PP2A by GW

Impact of the first wave of COVID-19 epidemy on the surgical management of sigmoid diverticular disease in France: National French retrospective study

International audienceObjective: To analyze the surgical management of sigmoid diverticular disease (SDD) before, during, and after the first containment rules (CR) for the first wave of COVID-19.Methods: From the French Surgical Association multicenter series, this study included all patients operated on between January 2018 and September 2021. Three groups were compared: A (before CR period: 01/01/18-03/16/20), B (CR period: 03/17/20-05/03/20), and C (post CR period: 05/04/20-09/30/21).Results: A total of 1965 patients (A n = 1517, B n = 52, C n = 396) were included. The A group had significantly more previous SDD compared to the two other groups (p = 0.007), especially complicated (p = 0.0004). The rate of peritonitis was significantly higher in the B (46.1%) and C (38.4%) groups compared to the A group (31.7%) (p = 0.034 and p = 0.014). As regards surgical treatment, Hartmann's procedure was more often performed in the B group (44.2%, vs A 25.5% and C 26.8%, p = 0.01). Mortality at 90 days was significantly higher in the B group (9.6%, vs A 4% and C 6.3%, p = 0.034). This difference was also significant between the A and B groups (p = 0.048), as well as between the A and C groups (p = 0.05). There was no significant difference between the three groups in terms of postoperative morbidity.Conclusion: This study shows that the management of SDD was impacted by COVID-19 at CR, but also after and until September 2021, both on the initial clinical presentation and on postoperative mortality

Rapid Improvement after Starting Elexacaftor–Tezacaftor–Ivacaftor in Patients with Cystic Fibrosis and Advanced Pulmonary Disease

International audienceRationale: Elexacaftor-tezacaftor-ivacaftor is a CFTR (cystic fibrosis [CF] transmembrane conductance regulator) modulator combination, developed for patients with CF with at least one Phe508del mutation. Objectives: To evaluate the effects of elexacaftor-tezacaftor- ivacaftor in patients with CF and advanced respiratory disease. Methods: A prospective observational study, including all patients aged ⩾12 years and with a percent-predicted FEV1 (ppFEV1) <40 who initiated elexacaftor-tezacaftor-ivacaftor from December 2019 to August 2020 in France was conducted. Clinical characteristics were collected at initiation and at 1 and 3 months. Safety and effectiveness were evaluated by September 2020. National-level transplantation and mortality figures for 2020 were obtained from the French CF and transplant centers and registries. Measurements and Main Results: Elexacaftor-tezacaftor- ivacaftor was initiated in 245 patients with a median (interquartile range) ppFEV1 = 29 (24-34). The mean (95% confidence interval) absolute increase in the ppFEV1 was +15.1 (+13.8 to +16.4; P < 0.0001), and the mean (95% confidence interval) in weight was +4.2 kg (+3.9 to +4.6; P < 0.0001). The number of patients requiring long-term oxygen, noninvasive ventilation, and/or enteral tube feeding decreased by 50%, 30%, and 50%, respectively (P < 0.01). Although 16 patients were on the transplant waiting list and 37 were undergoing transplantation evaluation at treatment initiation, only 2 received a transplant, and 1 died. By September 2020, only five patients were still on the transplantation path. Compared with the previous 2 years, a twofold decrease in the number of lung transplantations in patients with CF was observed in 2020, whereas the number of deaths without transplantation remained stable. Conclusions: In patients with advanced disease, elexacaftor-tezacaftor-ivacaftor is associated with rapid clinical improvement, often leading to the indication for lung transplantation being suspended