Dosimetric and geometric end-to-end accuracy of a magnetic resonance guided linear accelerator.

The introduction of real-time imaging by magnetic resonance guided linear accelerators (MR-Linacs) enabled adaptive treatments and gating on the tumor position. Different end-to-end tests monitored the accuracy of our MR-Linac during the first year of clinical operation. We report on the stability of these tests covering a static, adaptive and gating workflow. Film measurements showed gamma passing rates of 96.4% ± 3.4% for the static tests (five measurements) and for the two adaptive tests 98.9% and 99.99%, respectively (criterion 2%/2mm). The gated point dose measurements in the breathing phantom were 2.7% lower than in the static phantom.

CLASP2 binding to curved microtubule tips promotes flux and stabilizes kinetochore attachments.

CLASPs are conserved microtubule plus-end-tracking proteins that suppress microtubule catastrophes and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore-microtubule dynamics required for chromosome segregation fidelity, but the underlying mechanism remains unknown. Here, we found that human CLASP2 exists predominantly as a monomer in solution, but it can self-associate through its C-terminal kinetochore-binding domain. Kinetochore localization was independent of self-association, and driving monomeric CLASP2 to kinetochores fully rescued normal kinetochore-microtubule dynamics, while partially sustaining mitosis. CLASP2 kinetochore localization, recognition of growing microtubule plus-ends through EB-protein interaction, and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains independently sustained normal spindle length, timely spindle assembly checkpoint satisfaction, chromosome congression, and faithful segregation. Measurements of kinetochore-microtubule half-life and poleward flux revealed that CLASP2 regulates kinetochore-microtubule dynamics by integrating distinctive microtubule-binding properties at the kinetochore-microtubule interface. We propose that kinetochore CLASP2 suppresses microtubule depolymerization and detachment by binding to curved protofilaments at microtubule plus-ends.

Asociación entre niveles de proteína C-reactiva y óxido nítrico con el pronóstico de pacientes con infarto cerebral agudo / Association of C-reactive protein and nitric oxide levels with the prognosis of acute ischemic stroke

Introducción: La inflamación y alteraciones en la biodisponibilidad del óxido nítrico (NO) han sido involucradas en la fisiopatología de la enfermedad cerebrovascular. Objetivo: El objetivo del estudio fue determinar el valor pronóstico de la medición de metabolitos del NO y marcadores inflamatorios en pacientes con infarto cerebral agudo. Materiales y métodos: Se incluyeron 158 pacientes con diagnóstico de infarto cerebral agudo en un estudio observacional de cohorte. Entre 48 y 72 horas del inicio de los síntomas se tomó una muestra de sangre para determinación del perfil bioquímico, marcadores inflamatorios (PCR, IL1-β, IL6, TNF-α) y nitritos/nitratos plasmáticos. Se realizó seguimiento durante 2 años para determinar la aparición de un nuevo evento (infarto cerebral, infarto de miocardio, falla cardiaca) o muerte de origen vascular. Un análisis de regresión multivariada de Cox permitió determinar los factores asociados independientemente con el desenlace. Resultados: La edad promedio fue 70,5 ± 12,8 años. El 39,2% de los sujetos incluidos presentó el desenlace durante los primeros 24 meses de seguimiento. Los niveles de PCR > 12 mg/L (HR 2,22, IC 95% 1,07-4,59) y el puntaje > 13 en la escala NIHSS al ingreso (HR 2,81 IC 95% 1,46-5,41) se encontraron significativamente asociados con mayor riesgo de presentar un nuevo evento. La combinación de niveles de PCR < 12 mg/L y nitritos/nitratos < 35,5 µmol/L se identificó como un factor protector (HR 0,21, IC 95% 0,06-0,71). Conclusión: Este estudio sugiere que la determinación de nitritos/nitratos en conjunto con los niveles de PCR puede ser de utilidad para estratificar el riesgo de nuevos eventos en pacientes con infarto cerebral agudo.

Introduction: Inflammation and alterations in the bioavailability of nitric oxide (NO) have been involved in the pathophysiology of cerebrovascular disease. Objective: The aim of the study was to determine the prognostic value of measuring NO metabolites and inflammatory markers in patients with acute ischemic stroke. Materials and methods: A total of 158 patients with acute ischemic stroke were included in an observational cohort study. Between 48 and 72 hours post admission, a fasting blood sample was taken to determine the biochemical profile, inflammatory markers (CRP, IL1-β, IL6, TNF-α) and nitrites/nitrates plasma levels. The cohort's follow-up was conducted for two years to determine the occurrence of a new event (stroke, myocardial infarction, heart failure) or death of vascular origin. Comparisons between groups were made using the log-rank test. A Cox multivariate regression analysis permitted to determine factors independently associated with the outcome. Result: The mean age was 70.5 ± 12.8 years. 39.2% of the subjects presented the outcome during the first 24 months of follow-up. CRP levels > 12 mg/L (HR 2.22, 95% CI 1.07-4.59) and a score > 13 on the NIHSS scale at admission (HR 2.81 95% CI 1.46-5.41) were significantly associated with an increased risk of a new event. The combination of CRP levels < 12 mg/L and nitrites/nitrates levels < 35.5 mmol/L was identified as a protective factor (HR 0.21, 95% CI 0.06-0.71). Conclusion: This study demonstrates that the determination of CRP and NOx levels could be beneficial in clinical practice to stratify the risk of future events or death of vascular origin in acute ischemic stroke patients.

Cdk1 and Plk1 mediate a CLASP2 phospho-switch that stabilizes kinetochore-microtubule attachments.

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)-microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT-MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs was enhanced by CLASP2 phosphorylation on S1234. This was specifically required to stabilize KT-MT attachments important for chromosome alignment and to coordinate KT and non-KT MT dynamics necessary to maintain spindle bipolarity. CLASP2 C-terminal phosphorylation by Plk1 was also required for chromosome alignment and timely satisfaction of the SAC. We propose that Cdk1 and Plk1 mediate a fine CLASP2 "phospho-switch" that temporally regulates KT-MT attachment stability.

The fission yeast rDNA-binding protein Reb1 regulates G1 phase under nutritional stress.

Yeast Reb1 and its mammalian ortholog TTF1 are conserved Myb-type DNA-binding proteins that bind to specific sites near the 3'-end of rRNA genes (rDNA). Here, they participate in the termination of transcription driven by RNA polymerase I and block DNA replication forks approaching in the opposite direction. We found that Schizosaccharomyces pombe Reb1 also upregulates transcription of the ste9(+) gene that is required for nitrogen-starvation-induced growth arrest with a G1 DNA content and sexual differentiation. Ste9 activates the anaphase-promoting complex or cyclosome ('APC/C') in G1, targeting B-cyclin for proteasomal degradation in response to nutritional stress. Reb1 binds in vivo and in vitro to a specific DNA sequence at the promoter of ste9(+), similar to the sequence recognized in the rDNA, and this binding is required for ste9(+) transcriptional activation and G1 arrest. This suggests that Reb1 acts as a link between rDNA metabolism and cell cycle control in response to nutritional stress. In agreement with this new role for Reb1 in the regulation of the G1-S transition, reb1Δ and wee1(ts) mutations are synthetically lethal owing to the inability of these cells to lengthen G1 before entering S phase. Similarly, reb1Δ cdc10(ts) cells are unable to arrest in G1 and die at the semi-permissive temperature.

A PI3K activity-independent function of p85 regulatory subunit in control of mammalian cytokinesis.

Cytosolic division in mitotic cells involves the function of a number of cytoskeletal proteins, whose coordination in the spatio-temporal control of cytokinesis is poorly defined. We studied the role of p85/p110 phosphoinositide kinase (PI3K) in mammalian cytokinesis. Deletion of the p85alpha regulatory subunit induced cell accumulation in telophase and appearance of binucleated cells, whereas inhibition of PI3K activity did not affect cytokinesis. Moreover, reconstitution of p85alpha-deficient cells with a Deltap85alpha mutant, which does not bind the catalytic subunit, corrected the cytokinesis defects of p85alpha(-/-) cells. We analyzed the mechanism by which p85alpha regulates cytokinesis; p85alpha deletion reduced Cdc42 activation in the cleavage furrow and septin 2 accumulation at this site. As Cdc42 deletion also triggered septin 2 and cytokinesis defects, a mechanism by which p85 controls cytokinesis is by regulating the local activation of Cdc42 in the cleavage furrow and in turn septin 2 localization. We show that p85 acts as a scaffold to bind Cdc42 and septin 2 simultaneously. p85 is thus involved in the spatial control of cytosolic division through regulation of Cdc42 and septin 2, in a PI3K-activity independent manner.

Phosphoinositide 3-kinase controls early and late events in mammalian cell division.

Phosphoinositide 3-kinase (PI3K) plays a crucial role in triggering cell division. To initiate this process, PI3K induces two distinct routes, of which one promotes cell growth and the other regulates cyclin-dependent kinases. Fine-tuned PI3K regulation is also required for later cell cycle phases. Here, we review the multiple points at which PI3K controls cell division and discuss its impact on human cancer.

Control of cyclin G2 mRNA expression by forkhead transcription factors: novel mechanism for cell cycle control by phosphoinositide 3-kinase and forkhead.

Cyclin G2 is an unconventional cyclin highly expressed in postmitotic cells. Unlike classical cyclins that promote cell cycle progression, cyclin G2 blocks cell cycle entry. Here we studied the mechanisms that regulate cyclin G2 mRNA expression during the cell cycle. Analysis of synchronized NIH 3T3 cell cultures showed elevated cyclin G2 mRNA expression levels at G(0), with a considerable reduction as cells enter cell cycle. Downregulation of cyclin G2 mRNA levels requires activation of phosphoinositide 3-kinase, suggesting that this enzyme controls cyclin G2 mRNA expression. Because the phosphoinositide 3-kinase pathway inhibits the FoxO family of forkhead transcription factors, we examined the involvement of these factors in the regulation of cyclin G2 expression. We show that active forms of the forkhead transcription factor FoxO3a (FKHRL1) increase cyclin G2 mRNA levels. Cyclin G2 has forkhead consensus motifs in its promoter, which are transactivated by constitutive active FoxO3a forms. Finally, interference with forkhead-mediated transcription by overexpression of an inactive form decreases cyclin G2 mRNA expression levels. These results show that FoxO genes regulate cyclin G2 expression, illustrating a new role for phosphoinositide 3-kinase and FoxO transcription factors in the control of cell cycle entry.