Two XMAP215/TOG Microtubule Polymerases, Alp14 and Dis1, Play Non-Exchangeable, Distinct Roles in Microtubule Organisation in Fission Yeast

Proper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOGmembers, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal “cut” phenotype. By implementing an artificial targeting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Interestingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.This work was supported by the Japan Society for the Promotion of Science (JSPS) (KAKENHI Scientific Research (A) 16H02503 and the Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (S2902) to T.T. and Scientific Research (C) 19K05813 to M.Y.)

Characterisation of putative class 1A DHODH-like proteins from Mucorales and dematiaceous mould species

Olorofim is a new antifungal in clinical development which has a novel mechanism of action against dihydroorotate dehydrogenase (DHODH). DHODH form a ubiquitous family of enzymes in the de novo pyrimidine biosynthetic pathway and are split into class 1A, class 1B and class 2. Olorofim specifically targets the fungal class 2 DHODH present in a range of pathogenic moulds. The nature and number of DHODH present in many fungal species have not been addressed for large clades of this kingdom. Mucorales species do not respond to olorofim; previous work suggests they have only class 1A DHODH and so lack the class 2 target that olorofim inhibits. The dematiaceous moulds have mixed susceptibility to olorofim, yet previous analyses imply that they have class 2 DHODH. As this is at odds with their intermediate susceptibility to olorofim, we hypothesised that these pathogens may maintain a second class of DHODH, facilitating pyrimidine biosynthesis in the presence of olorofim. The aim of this study was to investigate the DHODH repertoire of clinically relevant species of Mucorales and dematiaceous moulds to further characterise these pathogens and understand variations in olorofim susceptibility. Using bioinformatic analysis, S. cerevisiae complementation and biochemical assays of recombinant protein, we provide the first evidence that two representative members of the Mucorales have only class 1A DHODH, substantiating a lack of olorofim susceptibility. In contrast, bioinformatic analyses initially suggested that seven dematiaceous species appeared to harbour both class 1A-like and class 2-like DHODH genes. However, further experimental investigation of the putative class 1A-like genes through yeast complementation and biochemical assays characterised them as dihydrouracil oxidases rather than DHODHs. These data demonstrate variation in dematiaceous mould olorofim susceptibility is not due to a secondary DHODH and builds on the growing picture of fungal dihydrouracil oxidases as an example of horizontal gene transfer

Exploring the roles of kinesin-8 in mitotic progression

The mitotic spindle employs myriad proteins for chromosome segregation, in addition to monitoring the fidelity of this process and correcting mistakes before they become problematic for daughter cells. Key players in mitosis include the superfamily of kinesin motors, a variety of microtubule-associated proteins (MAPs) and members of the spindle assembly checkpoint; deregulation of these factors has been observed in multiple tumour types. Our interest lies in the conserved family of microtubule plus-end directed kinesin-8 motors, which have been implicated in some breast and colorectal cancers. Klp5/Klp6 constitute the S. pombe kinesin-8 complex, and like the human counterpart, Kif18A, localise dynamically on the spindle, interact with kinetochores and regulate microtubule dynamics to allow a timely and coordinated metaphaseanaphase transition. Although these functions have been assigned in multiple species, exact mechanisms of the diverse kinesin-8 functions have not been elucidated, particularly in fission yeast. In contrast to prior studies of Klp5/6, where mainly deletion or truncation mutants were used, we employ a targeted mutagenesis approach to look directly at kinesin-8 function and regulation. Though Klp5/6 are non-essential, we exploit their synthetic lethality with various MAPs to isolate temperature-sensitive alleles of klp5. We find that a specific mutation in the non-motor kinesin tail has a deletion-like phenotype, disrupting chromosome congression, bipolar kinetochore attachment, negative regulation of spindle length and overall control of microtubule dynamics. Additionally, the processivity of the mutant is greatly impaired in vitro. Using this loss-of-function mutant with the deletion of XMAP215/TOG family microtubule polymerase, dis1Δ, we find that both kinesin-8 and Dis1 synergistically negatively regulate pre-anaphase and anaphase A spindle elongation but promote bipolar kinetochore-microtubule attachments in distinct ways. The activity of both factors is required to prepare metaphase for a timely and concerted anaphase for high fidelity chromosome segregation

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42·4% vs 44·2%; absolute difference -1·69 [-9·58 to 6·11] p=0·67; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5-8] vs 6 [5-8] cm H2O; p=0·0011). ICU mortality was higher in MICs than in HICs (30·5% vs 19·9%; p=0·0004; adjusted effect 16·41% [95% CI 9·52-23·52]; p<0·0001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0·80 [95% CI 0·75-0·86]; p<0·0001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status

Search for Scalar Diphoton Resonances in the Mass Range 65−60065-600 GeV with the ATLAS Detector in pppp Collision Data at s\sqrt{s} = 8 TeVTeV

A search for scalar particles decaying via narrow resonances into two photons in the mass range 65–600 GeV is performed using $20.3\text{}\text{}{\mathrm{fb}}^{-1}$ of $\sqrt{s}=8\text{}\text{}\mathrm{TeV}$ $pp$ collision data collected with the ATLAS detector at the Large Hadron Collider. The recently discovered Higgs boson is treated as a background. No significant evidence for an additional signal is observed. The results are presented as limits at the 95% confidence level on the production cross section of a scalar boson times branching ratio into two photons, in a fiducial volume where the reconstruction efficiency is approximately independent of the event topology. The upper limits set extend over a considerably wider mass range than previous searches