Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex

BACKGROUND: In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus. RESULTS: Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene). CONCLUSIONS: We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Mortality of emergency abdominal surgery in high-, middle- and low-income countries

Background: Surgical mortality data are collected routinely in high-income countries, yet virtually no low- or middle-income countries have outcome surveillance in place. The aim was prospectively to collect worldwide mortality data following emergency abdominal surgery, comparing findings across countries with a low, middle or high Human Development Index (HDI). Methods: This was a prospective, multicentre, cohort study. Self-selected hospitals performing emergency surgery submitted prespecified data for consecutive patients from at least one 2-week interval during July to December 2014. Postoperative mortality was analysed by hierarchical multivariable logistic regression. Results: Data were obtained for 10 745 patients from 357 centres in 58 countries; 6538 were from high-, 2889 from middle- and 1318 from low-HDI settings. The overall mortality rate was 1⋅6 per cent at 24 h (high 1⋅1 per cent, middle 1⋅9 per cent, low 3⋅4 per cent; P < 0⋅001), increasing to 5⋅4 per cent by 30 days (high 4⋅5 per cent, middle 6⋅0 per cent, low 8⋅6 per cent; P < 0⋅001). Of the 578 patients who died, 404 (69⋅9 per cent) did so between 24 h and 30 days following surgery (high 74⋅2 per cent, middle 68⋅8 per cent, low 60⋅5 per cent). After adjustment, 30-day mortality remained higher in middle-income (odds ratio (OR) 2⋅78, 95 per cent c.i. 1⋅84 to 4⋅20) and low-income (OR 2⋅97, 1⋅84 to 4⋅81) countries. Surgical safety checklist use was less frequent in low- and middle-income countries, but when used was associated with reduced mortality at 30 days. Conclusion: Mortality is three times higher in low- compared with high-HDI countries even when adjusted for prognostic factors. Patient safety factors may have an important role. Registration number: NCT02179112 (http://www.clinicaltrials.gov)

The Role of Net1 Phosphorylation in Regulating CDC14 Release During Mitotic Exit

Exit from mitosis is as an important phase in the cell cycle. The molecular event that triggers the cell cycle transition from anaphase into the G1 state involves the inactivation of the cyclin-dependent kinase complex (Cdk) through multiple mechanisms that lead to both destruction of the cyclin subunit co-activator and direct Cdk kinase inhibition. These multiple mechanisms indicate the importance of regulating the inactivation of Cdk to ensure proper cell cycle progression and cytokinesis. We set out to examine the regulation of the protein phosphatase Cdc14. Cdc14 is thought to act through reversal of phosphorylation on key Cdk substrates that promote mitotic exit by stimulating the destruction and inactivation of Cdk. In Saccharomyces cerevisiae, activation of Cdc14 is achieved via release from its nucleolar inhibitor Net1/Cfi1. This activation is correlated with multi-site phosphorylation of Net1 in cells where Cdc14 appears to be released from the nucleolus. We set out to identify new components of the nucleolar complex known as RENT (Regulator of Nucleolar Silencing and Teleophase) which holds Cdc14 in an inactive state. This led to the identification of Casein Kinase II (CKII) as a new component of RENT. CKII was verified to co-immunoprecipitate with Net1; and mutants in CKII arrest in anaphase with unreleased Cdc14 and unsegregated rDNA. Interestingly, phospho-peptide mapping experiments from in vivo Net1 samples revealed phosphorylation of a CKII consensus sequence within Net1. In vivo mapping also revealed another subset of sites that matched the consensus sequence established for Cdk phosphorylation. Mutational analysis of these sites unveiled their involvement in Cdc14 release during early anaphase and a role for a network of genetically interacting proteins involved in Fourteen Early Anaphase Release (FEAR) in promoting these phosphorylations. In summary, the regulation of Cdc14 release via phosphorylation of its nucleolar inhibitor Net1 as demonstrated by this work highlights the importance of nucleolar sequesteration and regulated release as a mechanism of controlling important cell cycle factors and events. It also points to a fascinating role for Cdk in insuring its own destruction at the end of the cell cycle, thus promoting transition back into the G1 state.</p

Biotechnological aspects and pharmaceutical applications of bacterials proteases

Proteases enzymes are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms. Bacterial proteases enzymes have great pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. New technologies for rationally protein engineering proteases, as well as improved delivery options, will expand greatly the potential pharmaceutical applications of enzymes. Proteases are extensively applied agents in several sectors of pharmaceutical industry. Furthermore, numerous research applications predominant use of proteases has been in killing tumor cells, they are also emerging as useful agents in the treatment of digestive disorders, inflammation, and other diseases. The aim of this paper is to review the biotechnological aspects of proteases enzymes and summarize their pharmaceutical applications in the life sciences

Phosphorylation by cyclin B-Cdk underlies release of mitotic exit activator Cdc14 from the nucleolus

Budding yeast protein phosphatase Cdc14 is sequestered in the nucleolus in an inactive state during interphase by the anchor protein Net1. Upon entry into anaphase, the Cdc14 early anaphase release (FEAR) network initiates dispersal of active Cdc14 throughout the cell. We report that the FEARnetwork promotes phosphorylation of Net1 by cyclin-dependent kinase (Cdk) complexed with cyclin B1 or cyclin B2. These phosphorylations appear to be required for FEAR and sustain the proper timing of late mitotic events. Thus, a regulatory circuit exists to ensure that the arbiter of the mitotic state, Cdk, sets in motion events that culminate in exit from mitosis