How are Dynamic Microtubules Stably Tethered to Human Chromosomes?

During cell division, microtubules capture and pull chromosomes apart into two equal sets. Without the establishment of proper chromosome-microtubule attachment, microtubules cannot impart the pulling forces needed to separate sister chromatid pairs. How are chromosomes captured along microtubule walls? How is the attachment of chromosomes to dynamic microtubule-ends achieved and monitored? We discuss these key questions by considering the roles of kinetochore-bound microtubule regulating proteins and also the complex regulatory loops of kinases and phosphatases that control chromosome-microtubule attachment and ensure the accurate segregation of chromosomes

Role of phosphatases in the end-on conversion process

Proper attachment of chromosomes to microtubules is important for the accurate segregation of chromosomes and genome stability. The initial engagement of chromosomes happens along the lateral wall of microtubules through a highly specialised protein structure assembled on the centromeric DNA, the kinetochore. Ultimately, kinetochores must be attached to the ends of microtubules (a geometry called end- on attachment). A series of highly dynamic steps called the end-on conversion process, converts the initial immature lateral attachments into mature end-on attachments. How this process is finely tuned by phosphorylation and dephosphorylation to achieve stable attachments is still unclear. Furthermore, what is the role of microtubule-associated proteins in the stabilisation of kinetochore-microtubule attachments is unknown. This project aimed to study the role of phosphatases in the regulation of the end-on conversion process. First, I investigated the different contribution of the two outer-kinetochore phosphatases - BubR1- recruited PP2A-B56 and KNL1-recruited PP1 - in counteracting Aurora B kinase during the end-on conversion process. I found that BubR1-recruited PP2A-B56 plays an essential role in the process, but KNL1-recruited PP1 does not. I also investigated whether the HEC1/Ndc80 N-tail is a critical substrate of Aurora B phosphorylation for the stabilisation of the end-on attachments. Using a phospho-dead mutant of the HEC1/Ndc80 N-tail, I discovered that cells are still susceptible to Aurora B activity, indicating downstream pathways independent of HEC1/Ndc80. Then, I studied the biological role of the Astrin C-terminus, where an evolutionarily conserved RVMF motif, a putative PP1 binding site, is located. My findings show C-terminal Astrin mutants fail to localise at kinetochores of both monopolar and bipolar spindles; induce defects in the end-on conversion process in monopolar spindles and prolong mitosis time with increased Mad2 levels at the outer-kinetochore. A kinase inhibitor assay showed that kinetochore-microtubule attachment defects in Astrin mutant expressing cells could be rescued when both Aurora B and Cdk1 kinases are inhibited, suggesting a role for Astrin’s C-terminus in counteracting Aurora B and Cdk1 activity. Finally, I probed the putative interaction of the Astrin C-terminus and PP1 using biochemistry, cell biology and fluorescence microscopy techniques. I discovered that artificially targeting PP1 onto the Astrin C-terminus but not on the N-terminus rescues mutants localisation defects at the kinetochore. In summary, my results indicate that Astrin and PP1 interact at the kinetochore of living cells. In conclusion, my work shows that mitotic phosphatases have distinctive contributions in the regulation of the dynamic steps of the end-on conversion process and that Astrin is a potential PP1 phosphatase recruiter at the outer-kinetochore, where is necessary for the stabilisation of end-on attachments.MRC DTP, CR UK, Department of Genetics, CCEI

Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation.

Defects in chromosome-microtubule attachment can cause chromosomal instability (CIN), frequently associated with infertility and aggressive cancers. Chromosome-microtubule attachment is mediated by a large macromolecular structure, the kinetochore. Sister kinetochores of each chromosome are pulled by microtubules from opposing spindle-poles, a state called biorientation which prevents chromosome missegregation. Kinetochore-microtubule attachments that lack the opposing-pull are detached by Aurora-B/Ipl1. It is unclear how mono-oriented attachments that precede biorientation are spared despite the lack of opposing-pull. Using an RNAi-screen, we uncover a unique role for the Astrin-SKAP complex in protecting mono-oriented attachments. We provide evidence of domains in the microtubule-end associated protein that sense changes specific to end-on kinetochore-microtubule attachments and assemble an outer-kinetochore crescent to stabilise attachments. We find that Astrin-PP1 and Cyclin-B-CDK1 pathways counteract each other to preserve mono-oriented attachments. Thus, CIN prevention pathways are not only surveying attachment defects but also actively recognising and stabilising mature attachments independent of biorientation

“In Less than No Time”: Feasibility of Rotational Thromboelastometry to Detect Anticoagulant Drugs Activity and to Guide Reversal Therapy

Anticoagulant drugs (i.e., unfractionated heparin, low-molecular-weight heparins, vitamin K antagonists, and direct oral anticoagulants) are widely employed in preventing and treating venous thromboembolism (VTE), in preventing arterial thromboembolism in nonvalvular atrial fibrillation (NVAF), and in treating acute coronary diseases early. In certain situations, such as bleeding, urgent invasive procedures, and surgical settings, the evaluation of anticoagulant levels and the monitoring of reversal therapy appear essential. Standard coagulation tests (i.e., activated partial thromboplastin time (aPTT) and prothrombin time (PT)) can be normal, and the turnaround time can be long. While the role of viscoelastic hemostatic assays (VHAs), such as rotational thromboelastometry (ROTEM), has successfully increased over the years in the management of bleeding and thrombotic complications, its usefulness in detecting anticoagulants and their reversal still appears unclear

A Case Series of Patients Who Underwent Laparoscopic Extraperitoneal Radical Prostatectomy with the Simultaneous Implant of a Penile Prosthesis: Focus on Penile Length Preservation

Purpose: There are many grey areas in the field of penile rehabilitation after radical prostatectomy (RP). The preservation of the full dimensions of the penis is an important consideration for improving patients’ compliance for the treatment. We present the first case series of patients treated by laparoscopic extraperitoneal RP and simultaneous penile prosthesis implantation (PPI) in order to preserve the full length of the penis and to improve patients’ satisfaction. Materials and Methods: From June 2013 to June 2014, 10 patients underwent simultaneous PPI (with an AMS InhibiZone prosthesis) and RP. Patients were evaluated by means of urological visits, questionnaires, and objective measurements before surgery, at discharge from the hospital, on postoperative days 21 to 28, each 3 months for the first year, and each 6 months thereafter. The main outcome measures were biochemical recurrence-free rate, penile length, and quality of life. Results: Ten patients (mean age of 61 years; completed the study follow-up period (median, 32.2 months). No difference was found between the time of surgery and the 2-year follow-up evaluation in terms of penile length. The pre-surgery 36-Item Short Form Health Survey (SF-36) median score was 97. Patients were satisfied with their penile implants, and couples’ level of sexual satisfaction was rated median 8. The median postoperative SF-36 score was 99 at 3 months follow-up. Conclusions: Laparoscopic extraperitoneal RP surgery with simultaneous PPI placement seems to be an interesting possibility to propose to motivated patients for preserving the length of the penis and improving their satisfaction

The relationship between species and spectral diversity in grassland communities is mediated by their vertical complexity

Aims: The link between spectral diversity and in-\uadsitu plant biodiversity is one prom- ising approach to using remote sensing for biodiversity assessment. Nevertheless, there is little evidence as to whether this link is maintained at fine scales, as well as to how it is influenced by vegetation's vertical complexity. Here we test, at the com- munity level in grasslands, the link between diversity of the spectral signal (SDiv) and taxonomic diversity (TDiv), and the influence of vertical complexity. Methods: We used 196 1.5 m 7 1.5 m experimental communities with different biodiversity levels. To measure vertical complexity, we quantified height diversity (HDiv) of the most abundant species in the community. TDiv was calculated using the Shannon index based on species cover. Canopy spectral information was gathered using an unmanned aerial vehicle (UAV) mounted with a multi-\uadspectral sensor provid- ing spectral information via six 10-\uadnm bands covering the visible and near-\uadinfrared region at a spatial resolution of 3 cm. We measured SDiv in a core area of 1 m 71 m within the communities as mean Euclidean distance of all pixels in a feature space spanned between the two first components of a PCA calculated for the complete raster stack. We modelled SDiv through mixed-\uadeffect linear models, using TDiv, HDiv, and their interaction as fixed-\uadeffect predictors. Results: Contrary to our expectations, TDiv was negatively linked to SDiv. The diversity in plant height was positively related to SDiv. More importantly, diversity in plant height and TDiv had a significant negative interaction, meaning the more complex the vegeta- tion was in terms of height, the more the SDiv \u2013\uad TDiv relationship became negative. Conclusions: Our results suggest that in order to exploit the SDiv\u2013\uad TDiv link for moni- toring purposes, it needs to be contextualized. Moreover, the results highlight that communities\u2019 functional characteristics (i.e. plant height) mediate such a link, calling for new insights into the relation between SDiv and functional diversity

Cortical propagation tracks functional recovery after stroke.

Stroke is a debilitating condition affecting millions of people worldwide. The development of improved rehabilitation therapies rests on finding biomarkers suitable for tracking functional damage and recovery. To achieve this goal, we perform a spatiotemporal analysis of cortical activity obtained by wide-field calcium images in mice before and after stroke. We compare spontaneous recovery with three different post-stroke rehabilitation paradigms, motor training alone, pharmacological contralesional inactivation and both combined. We identify three novel indicators that are able to track how movement-evoked global activation patterns are impaired by stroke and evolve during rehabilitation: the duration, the smoothness, and the angle of individual propagation events. Results show that, compared to pre-stroke conditions, propagation of cortical activity in the subacute phase right after stroke is slowed down and more irregular. When comparing rehabilitation paradigms, we find that mice treated with both motor training and pharmacological intervention, the only group associated with generalized recovery, manifest new propagation patterns, that are even faster and smoother than before the stroke. In conclusion, our new spatiotemporal propagation indicators could represent promising biomarkers that are able to uncover neural correlates not only of motor deficits caused by stroke but also of functional recovery during rehabilitation. In turn, these insights could pave the way towards more targeted post-stroke therapies

Cannabidiol in the acute phase of febrile infection‐related epilepsy syndrome (FIRES)

Abstract Febrile infection‐related epilepsy syndrome (FIRES) is a prolonged refractory status epilepticus (SE) that develops among healthy individuals after a febrile infection. FIRES treatment is challenging due to its poor response to antiseizure medications (ASMs) and anesthetic drugs. The use of cannabidiol (CBD) as an adjunctive treatment has been suggested, albeit data about its role in the acute phase is lacking. This report describes the use of purified CBD in the acute phase of two pediatric cases of FIRES and their long‐term outcome. Both children were treated with several ASMs, immunomodulators, anesthetics, and nonpharmacological treatment (ketogenic diet). CBD was administered, as an adjunctive treatment, through nasogastric tube about 30 days after onset. SE resolved within 3 days of reaching the target dose and both were seizure‐free for 1 year after. Although it is difficult to define the extent to which each previous therapy contributed to recovery, in both cases CBD therapy was a turning point, reinforcing its potential role as add‐on treatment in the acute phase of FIRES