Dependency of the Spindle Assembly Checkpoint on Cdk1 Renders the Anaphase Transition Irreversible

SummaryActivation of anaphase-promoting complex/cyclosome (APC/CCdc20) by Cdc20 is delayed by the spindle assembly checkpoint (SAC). When all kinetochores come under tension, the SAC is turned off and APC/CCdc20 degrades cyclin B and securin, which activates separase [1]. The latter then cleaves cohesin holding sister chromatids together [2]. Because cohesin cleavage also destroys the tension responsible for turning off the SAC, cells must possess a mechanism to prevent SAC reactivation during anaphase, which could be conferred by a dependence of the SAC on Cdk1 [3–5]. To test this, we analyzed mouse oocytes and embryos expressing nondegradable cyclin B together with a Cdk1-resistant form of separase. After biorientation and SAC inactivation, APC/CCdc20 activates separase but the resulting loss of (some) cohesion is accompanied by SAC reactivation and APC/CCdc20 inhibition, which aborts the process of further securin degradation. Cyclin B is therefore the only APC/CCdc20 substrate whose degradation at the onset of anaphase is necessary to prevent SAC reactivation. The mutual activation of tension sensitive SAC and Cdk1 creates a bistable system that ensures complete activation of separase and total downregulation of Cdk1 when all chromosomes have bioriented

APC/C(Cdh1) Enables Removal of Shugoshin-2 from the Arms of Bivalent Chromosomes by Moderating Cyclin-Dependent Kinase Activity

In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/C(Cdc20)) activates separase and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2, sister chromatids remain attached through centromeric/pericentromeric cohesin. We show here that, by promoting proteolysis of cyclins and Cdc25B at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/C(Cdh1)) delays the increase in Cdk1 activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/C(Cdh1) creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase, an event crucial for the efficient resolution of chiasmata

Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes

This article is distributed under the terms of the Creative Commons Attribution License.-- et al.Accurate chromosome segregation depends on coordination between cohesion resolution and kinetochore-microtubule interactions (K-fibers), a process regulated by the spindle assembly checkpoint (SAC). How these diverse processes are coordinated remains unclear. We show that in mammalian oocytes Shugoshin-like protein 2 (Sgol2) in addition to protecting cohesin, plays an important role in turning off the SAC, in promoting the congression and bi-orientation of bivalents on meiosis I spindles, in facilitating formation of K-fibers and in limiting bivalent stretching. Sgol2's ability to protect cohesin depends on its interaction with PP2A, as is its ability to silence the SAC, with the latter being mediated by direct binding to Mad2. In contrast, its effect on bivalent stretching and K-fiber formation is independent of PP2A and mediated by recruitment of MCAK and inhibition of Aurora C kinase activity respectively. By virtue of its multiple interactions, Sgol2 links many of the processes essential for faithful chromosome segregation.AR was supported by a PhD fellowship from the Boehringer Ingelheim Fonds. BM and OS were supported by a grant of the Deutsche Forschungsgemeinschaft (STE997/3-2 within the priority program SPP1384). AP was supported by SAF2011-25252. This study was funded by the European Community’s Seventh Framework MitoSys/241548, Medical Research Council and Wellcome Trust and Ministerio de Ciencia e Innovacion SAF2011-25252.Peer Reviewe

Regulation of anaphase in mammalian meiosis

Missegregation of chromosomes during meiosis leads to formation of aneuploid eggs. Estimates suggest that in humans, about 10-30% of fertilised eggs and one-third of all miscarriages are aneuploid. Accurate chromosome segregation depends on the coordination between stepwise cohesion resolution and attachments of homologous chromosomes through kinetochores to microtubules, emanating from opposite poles of the cell. The Spindle Assembly Checkpoint (SAC) monitors microtubule-kinetochore attachments and prevents resolution of cohesin complexes by inhibiting the ubiquitin ligase APC/CCdc20 until all aberrant microtubule-kinetochore attachments have been rectified by an Aurora Kinase-dependent error correction machinery. During meiosis, these pathways work in seamless coordination to achieve balanced segregation of the genome at the first meiotic division.The cross-talk between different cell cycle pathways requires members with shared affiliations. During my DPhil studies, I worked on understanding the role of two such proteins, namely Bub1 (budding uninhibited by benzimidazoles 1) and Sgol2 (Shugoshin-like protein 2) in mouse oocytes.During the first meiotic division, Bub1 maintains the SAC, and through its kinase activity, Bub1 recruits Sgol2 to kinetochores to protect centromeric cohesion. This recruitment is essential for two rounds of chromosomes segregation in meiosis. Thus, Bub1 localisation at kinetochores can coordinate the timing of anaphase with the centromeric cohesion protection.During the first meiotic division, Sgol2 protects centromeric cohesion by recruiting PP2A to kinetochores, accelerates cohesin resolution by silencing the SAC through its interaction with PP2A and Mad2, and also promotes biorientation and congression of homologous chromosomes by its interaction with MCAK and through dephosphorylation of Aurora B/C kinase substrates at kinetochores.This research revealed that Bub1 and Sgol2 can regulate anaphase by linking multiple cell cycle pathways that work together to achieve faithful chromosomes segregation in mammalian meiosis.</p

Regulation of anaphase in mammalian meiosis

Missegregation of chromosomes during meiosis leads to formation of aneuploid eggs. Estimates suggest that in humans, about 10-30% of fertilised eggs and one-third of all miscarriages are aneuploid. Accurate chromosome segregation depends on the coordination between stepwise cohesion resolution and attachments of homologous chromosomes through kinetochores to microtubules, emanating from opposite poles of the cell. The Spindle Assembly Checkpoint (SAC) monitors microtubule-kinetochore attachments and prevents resolution of cohesin complexes by inhibiting the ubiquitin ligase APC/CCdc20 until all aberrant microtubule-kinetochore attachments have been rectified by an Aurora Kinase-dependent error correction machinery. During meiosis, these pathways work in seamless coordination to achieve balanced segregation of the genome at the first meiotic division.The cross-talk between different cell cycle pathways requires members with shared affiliations. During my DPhil studies, I worked on understanding the role of two such proteins, namely Bub1 (budding uninhibited by benzimidazoles 1) and Sgol2 (Shugoshin-like protein 2) in mouse oocytes.During the first meiotic division, Bub1 maintains the SAC, and through its kinase activity, Bub1 recruits Sgol2 to kinetochores to protect centromeric cohesion. This recruitment is essential for two rounds of chromosomes segregation in meiosis. Thus, Bub1 localisation at kinetochores can coordinate the timing of anaphase with the centromeric cohesion protection.During the first meiotic division, Sgol2 protects centromeric cohesion by recruiting PP2A to kinetochores, accelerates cohesin resolution by silencing the SAC through its interaction with PP2A and Mad2, and also promotes biorientation and congression of homologous chromosomes by its interaction with MCAK and through dephosphorylation of Aurora B/C kinase substrates at kinetochores.This research revealed that Bub1 and Sgol2 can regulate anaphase by linking multiple cell cycle pathways that work together to achieve faithful chromosomes segregation in mammalian meiosis.This thesis is not currently available via ORA

Identification of myeloperoxidase, alpha-defensin and calgranulin in calcium oxalate renal stones.

BACKGROUND: In order to understand the mechanism of stone genesis, it is essential to determine the characteristics of macromolecules constituting the urinary stones. We characterized proteins from the inner core and outer matrix of calcium oxalate (CaOx) renal stones. METHODS: Inner core and outer matrix of CaOx renal stones were separated and proteins were extracted with a buffer containing SDS and beta-mercaptoethanol. Proteins were analyzed and purified by SDS-PAGE and RP-HPLC respectively. The protein bands from gel and protein fractions were sequenced by MALDI TOF mass spectrometry. ELISA, western and slot blot immunoassays were performed to confirm the identity of the proteins in stones and urine of the stone formers. The potential of the identified protein as an effective promoter or inhibitor was assessed by observing their effects on CaOx crystallization using aggregometer. RESULTS: The inner core extract predominantly exhibited protein species in the molecular weight range of 12-14 kDa. However, a 66 kDa band, identified as osteopontin was also detected in the inner core along with outer matrix and in the urine of stone formers and non stone formers. Purification of low molecular weight proteins was carried out by reversed phase HPLC. Tandem mass spectrometry analysis identified them asmyeloperoxidase chain A (MPO-A), alpha-defensin, and calgranulin. ELISA, western blot and slot-blot immuno-assays further confirmed their presence restricted to the inner core and not in the outer matrix. Turbidity assays showed that low molecular weight renal stone proteins promoted the aggregation of CaOx crystals. CONCLUSIONS: Persistent hyperoxaluria leads to tubular epithelial injury, resulting in the release of these anti-inflammatory proteins. These proteins could have been first adsorbed on CaOx crystals thereby become a part of nucleation process leading to inner matrix formation

Effect of demographic and clinical characteristics of open wound pressure ulcers in patients at tertiary care hospital of Karachi

Pressure ulcers acquired in a hospital are a common problem in patients with neurological disorders. Although a number of risk factors have been identified, no relationships between risk profile characteristics and pressure ulcer outcomes in hospitalized patients in Karachi have been described. This research aimed to identify the elements that contributed to the emergence of open-wound pressure ulcers in patients with neurological disorders who were being treated in hospitals. A cross-sectional analytical study design was used to enroll 93 study participants from Pakistan's Murshid Hospital and Healthcare Centre (MHHCC). The data was collected using a structured questionnaire. The Chi-Square test and an independent t-test were used to analyze the data using SPSS 21 to determine the results. The study discovered that the Glasgow Comma Scale (p = 0.001), length of hospital stay (p = 0.001), and back care (p = 0.03) were statistically associated with the open wound pressure ulcer. In conclusion, hospitalized patients with neurological disorders showed a significant difference between open wound and close PU in terms of mean GCS, length of hospital stay, and back care.pressure ulcer (PU

Point prevalence probing of antimicrobial prescription patterns from a developing country

Irrational use of antibiotics intensifies resistance and jeopardizes advances made in modern medicine. We aimed to conduct a baseline gap analysis survey on antibiotic prescription practices across Pakistan. This multi-centered cross-sectional survey was conducted at six public sector tertiary care hospitals from February 2021 to March 2021. Data related to various variables including hospital infrastructure, policies and practices, monitoring and feedback, and epidemiological, clinical, and antibiotic prescription for surveyed patients was collected using World Health Organization (WHO) Point Prevalence Survey (PPS) methodology. In a survey of 837 inpatients, 78.5% were prescribed antibiotics. Most commonly prescribed antimicrobial was ceftriaxone (21.7%), followed by metronidazole (17.3%), cefoperazone-sulbactam (8.4%), amoxicillin-clavulanate (6.3%), and piperacillin/tazobactam (5.9%). Surgical prophylaxis (36.7%) and community-acquired infections (24.7%) were the main reasons for antibiotic prescriptions. Single antibiotics were given to 46.7% of patients, 39.9% received a combination of two antibiotics, and 12.5% were prescribed three or more antibiotics. Among six hospitals surveyed, two had drug and therapeutic committees, three had infection prevention and control committees, and one had an antibiotic formulary. Findings demonstrate high consumption of broad-spectrum antimicrobials and emphasize the importance of expanding antimicrobial stewardship programs among hospitals. Mentoring clinical teams could help rationalize antimicrobial use.</p

Das Ende neoliberaler Hegemonie durch soziale Bewegungen? (English translation: Reading Neoliberalism as a Social Movement from Above)

In this article we explore the relationship between Marxist theory and social movements, in particular how this relationship works in the specific historical period that we call the twilight of neoliberalism