Distinct Functions for Mammalian CLASP1 and-2 During Neurite and Axon Elongation

Mammalian cytoplasmic linker associated protein 1 and -2 (CLASP1 and -2) are microtubule (MT) plus-end tracking proteins that selectively stabilize MTs at the edge of cells and that promote MT nucleation and growth at the Golgi, thereby sustaining cell polarity. In vitro analysis has shown that CLASPs are MT growth promoting factors. To date, a single CLASP1 isoform (called CLASP1α) has been described, whereas three CLASP2 isoforms are known (CLASP2α, -β, and -γ). Although CLASP2β/γ are enriched in neurons, suggesting isoform-specific functions, it has been proposed that during neurite outgrowth CLASP1 and -2 act in a redundant fashion by modulating MT dynamics downstream of glycogen synthase kinase 3 (GSK3). Here, we show that in differentiating N1E-115 neuroblastoma cells CLASP1 and CLASP2 differ in their accumulation at MT plus-ends and display different sensitivity to GSK3-mediated phosphorylation, and hence regulation. More specifically, western blot (WB) analysis suggests that pharmacological inhibition of GSK3 affects CLASP2 but not CLASP1 phosphorylation and fluorescence-based microscopy data show that GSK3 inhibition leads to an increase in the number of CLASP2-decorated MT ends, as well as to increased CLASP2 staining of individual MT ends, whereas a reduction in the number of CLASP1-decorated ends is observed. Thus, in N1E-115 cells CLASP2 appears to be a prominent target of GSK3 while CLASP1 is less sensitive. Surprisingly, knockdown of either CLASP causes phosphorylation of GSK3, pointing to the existence of feedback loops between CLASPs and GSK3. In addition, CLASP2 depletion also leads to the activation of protein kinase C (PKC). We found that these differences correlate with opposite functions of CLASP1 and CLASP2 during neuronal differentiation, i.e., CLASP1 stimulates neurite extension, whereas CLASP2 inhibits it. Consistent with knockdown results in N1E-115 cells, primary Clasp2 knockout (KO) neurons exhibit early accelerated neurite and axon outgrowth, showing longer axons than control neurons. We propose a model in which neurite outgrowth is fine-tuned by differentially posttranslationally modified isoforms of CLASPs acting at distinct intracellular locations, thereby targeting MT stabilizing activities of the CLASPs and controlling feedback signaling towards upstream kinases. In summary, our findings provide new insight into the roles of neuronal CLASPs, which emerge as regulators acting in different signaling pathways and locally modulating MT behavior during neurite/axon outgrowth

Diagnostic accuracy of a clinical diagnosis of idiopathic pulmonary fibrosis: An international case-cohort study

We conducted an international study of idiopathic pulmonary fibrosis (IPF) diagnosis among a large group of physicians and compared their diagnostic performance to a panel of IPF experts. A total of 1141 respiratory physicians and 34 IPF experts participated. Participants evaluated 60 cases of interstitial lung disease (ILD) without interdisciplinary consultation. Diagnostic agreement was measured using the weighted kappa coefficient (\u3baw). Prognostic discrimination between IPF and other ILDs was used to validate diagnostic accuracy for first-choice diagnoses of IPF and were compared using the Cindex. A total of 404 physicians completed the study. Agreement for IPF diagnosis was higher among expert physicians (\u3baw=0.65, IQR 0.53-0.72, p20 years of experience (C-index=0.72, IQR 0.0-0.73, p=0.229) and non-university hospital physicians with more than 20 years of experience, attending weekly MDT meetings (C-index=0.72, IQR 0.70-0.72, p=0.052), did not differ significantly (p=0.229 and p=0.052 respectively) from the expert panel (C-index=0.74 IQR 0.72-0.75). Experienced respiratory physicians at university-based institutions diagnose IPF with similar prognostic accuracy to IPF experts. Regular MDT meeting attendance improves the prognostic accuracy of experienced non-university practitioners to levels achieved by IPF experts

Oxytocin moderates corticolimbic social stress reactivity in cocaine use disorder and healthy controls

Social stress can contribute to the development of substance use disorders (SUDs) and increase the likelihood of relapse. Oxytocin (OT) is a potential pharmacotherapy that may buffer the effects of social stress on arousal and reward neurocircuitry. However, more research is needed to understand how OT moderates the brain's response to social stress in SUDs. The present study examined the effect of intransasal OT (24 IU) versus placebo (PBO) on corticolimbic functional connectivity associated with acute social stress in individuals with cocaine use disorder (CUD; n = 67) and healthy controls (HC; n = 52). Psychophysiological interaction modeling used the left and right amygdala as seed regions with the left and right orbitofrontal and anterior cingulate cortex as a priori regions of interest. Moderators of the OT response included childhood trauma history and biological sex, which were examined in independent analyses. The main finding was that OT normalized corticolimbic connectivity (left amygdala-orbitofrontal and left amygdala-anterior cingulate) as a function of childhood trauma such that connectivity was different between trauma-present and trauma-absent groups on PBO, but not between trauma groups on OT. Effects of OT on corticolimbic connectivity were not different as a function of diagnosis (CUD vs HC) or sex. However, OT reduced subjective anxiety during social stress for CUD participants who reported childhood trauma compared to PBO and normalized craving response as a function of sex in CUD. The present findings add to some prior findings of normalizing effects of OT on corticolimbic circuitry in individuals with trauma histories and provide some initial support that OT can normalize subjective anxiety and craving in CUD

The chaperonin CCT controls T cell receptor–driven 3D configuration of centrioles

© 2020 The Authors.T lymphocyte activation requires the formation of immune synapses (IS) with antigen-presenting cells. The dynamics of membrane receptors, signaling scaffolds, microfilaments, and microtubules at the IS determine the potency of T cell activation and subsequent immune response. Here, we show that the cytosolic chaperonin CCT (chaperonin-containing TCP1) controls the changes in reciprocal orientation of the centrioles and polarization of the tubulin dynamics induced by T cell receptor in T lymphocytes forming an IS. CCT also controls the mitochondrial ultrastructure and the metabolic status of T cells, regulating the de novo synthesis of tubulin as well as posttranslational modifications (poly-glutamylation, acetylation, Δ1 and Δ2) of αβ-tubulin heterodimers, fine-tuning tubulin dynamics. These changes ultimately determine the function and organization of the centrioles, as shown by three-dimensional reconstruction of resting and stimulated primary T cells using cryo-soft x-ray tomography. Through this mechanism, CCT governs T cell activation and polarity.Cryo-SXT work was supported by ALBA Synchrotron standard proposals 2015021148 and 2016021638 to F.J.C., N.B.M.-C., and J.M.V. This study was supported by grants SAF2017-82886-R (to F.S.-M.), PID2019-105872GB I00/AEI/10.13039/501100011033 (AEI/FEDER, UE), BFU2016-75984 (to J.M.V.), and BIO2015-67580-P and PGC2018-097019-B-I00 (to J.V.) from the Spanish Ministry of Economy and Competitiveness (MINECO), grants INFLAMUNE-S2017/BMD-23671 (to F.S.-M.) and P2018/NMT-4389 (to J.M.V.) from the Comunidad de Madrid, ERC-2011-AdG 294340-GENTRIS (to F.S.-M.), a 2019 grant from the Ramón Areces Foundation “Ciencias de la Vida y la Salud” and a 2018 grant from Ayudas Fundación BBVA a Equipos de Investigación Científica (to F.S.-M.), and grants PRB3 (IPT17/0019-ISCIII-SGEFI/ERDF), the Fundació Marató TV3 (grant 122/C/2015), and “La Caixa” Banking Foundation (HR17-00016 to FSM and HR17-00247 to J.V.). D.T. is supported by a PhD fellowship from La Caixa Foundation. Work in the Vernos lab was supported by the grant CSD2006-00023 from the Spanish Ministry of Science and Innovation and grants BFU2012-37163 and BFU2015-68726-P from the Spanish Ministry of Economy and Competitiveness. The CRG acknowledges support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme/Generalitat de Catalunya. CIBER Cardiovascular (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). The Centro Nacional de Biotecnología (CNB) is a Severo Ochoa Center of Excellence (MINECO award SEV 2017-0712)

Analysis of chromosomal polymorphism in barley (Hordeum vulgare L. ssp. vulgare ) and between H. vulgare and H. chilense using three-color fluorescence in situ hybridization (FISH)

The aim of the present work was to study chromosomal polymorphism within cultivated barley (Hordeum vulgare ssp. vulgare) using three-color fluorescence in situ hybridization (FISH). The physical distribution of the most frequently used, highly repetitive DNA sequences (GAA)7 specific for pericentromeric heterochromatic regions, the ribosomal DNA clone pTa71, specific for the 45S rDNA, and the barley-specific telomere-associated sequence HvT01, was investigated to reveal genetic diversity in metaphase spreads of ten barley genotypes with diverse geographical origin, growth habit and row number. A wild relative of barley, Hordeum chilense was also studied in order to compare the polymorphism between and within Hordeum species. Significant differences in the hybridization patterns of all three DNA probes could be detected between the two related species, but only probes pTa71 and HvT01 showed variation in the intensity and/or position of hybridization sites among genotypes of H. vulgare ssp. vulgare. The extent of polymorphism was less than that earlier reported for molecular markers and was restricted to the long chromosome arms, with differences between the chromosomes. 1H and 3H proved to be the most variable chromosomes and 4H and 6H the most conserved

Prevalence and risk factors for delirium in critically ill patients with COVID-19 (COVID-D): a multicentre cohort study

Background: To date, 750 000 patients with COVID-19 worldwide have required mechanical ventilation and thus are at high risk of acute brain dysfunction (coma and delirium). We aimed to investigate the prevalence of delirium and coma, and risk factors for delirium in critically ill patients with COVID-19, to aid the development of strategies to mitigate delirium and associated sequelae. Methods: This multicentre cohort study included 69 adult intensive care units (ICUs), across 14 countries. We included all patients (aged 6518 years) admitted to participating ICUs with severe acute respiratory syndrome coronavirus 2 infection before April 28, 2020. Patients who were moribund or had life-support measures withdrawn within 24 h of ICU admission, prisoners, patients with pre-existing mental illness, neurodegenerative disorders, congenital or acquired brain damage, hepatic coma, drug overdose, suicide attempt, or those who were blind or deaf were excluded. We collected de-identified data from electronic health records on patient demographics, delirium and coma assessments, and management strategies for a 21-day period. Additional data on ventilator support, ICU length of stay, and vital status was collected for a 28-day period. The primary outcome was to determine the prevalence of delirium and coma and to investigate any associated risk factors associated with development of delirium the next day. We also investigated predictors of number of days alive without delirium or coma. These outcomes were investigated using multivariable regression. Findings: Between Jan 20 and April 28, 2020, 4530 patients with COVID-19 were admitted to 69 ICUs, of whom 2088 patients were included in the study cohort. The median age of patients was 64 years (IQR 54 to 71) with a median Simplified Acute Physiology Score (SAPS) II of 40\ub70 (30\ub70 to 53\ub70). 1397 (66\ub79%) of 2088 patients were invasively mechanically ventilated on the day of ICU admission and 1827 (87\ub75%) were invasively mechanical ventilated at some point during hospitalisation. Infusion with sedatives while on mechanical ventilation was common: 1337 (64\ub70%) of 2088 patients were given benzodiazepines for a median of 7\ub70 days (4\ub70 to 12\ub70) and 1481 (70\ub79%) were given propofol for a median of 7\ub70 days (4\ub70 to 11\ub70). Median Richmond Agitation\u2013Sedation Scale score while on invasive mechanical ventilation was \u20134 (\u20135 to \u20133). 1704 (81\ub76%) of 2088 patients were comatose for a median of 10\ub70 days (6\ub70 to 15\ub70) and 1147 (54\ub79%) were delirious for a median of 3\ub70 days (2\ub70 to 6\ub70). Mechanical ventilation, use of restraints, and benzodiazepine, opioid, and vasopressor infusions, and antipsychotics were each associated with a higher risk of delirium the next day (all p 640\ub704), whereas family visitation (in person or virtual) was associated with a lower risk of delirium (p<0\ub70001). During the 21-day study period, patients were alive without delirium or coma for a median of 5\ub70 days (0\ub70 to 14\ub70). At baseline, older age, higher SAPS II scores, male sex, smoking or alcohol abuse, use of vasopressors on day 1, and invasive mechanical ventilation on day 1 were independently associated with fewer days alive and free of delirium and coma (all p<0\ub701). 601 (28\ub78%) of 2088 patients died within 28 days of admission, with most of those deaths occurring in the ICU. Interpretation: Acute brain dysfunction was highly prevalent and prolonged in critically ill patients with COVID-19. Benzodiazepine use and lack of family visitation were identified as modifiable risk factors for delirium, and thus these data present an opportunity to reduce acute brain dysfunction in patients with COVID-19. Funding: None. Translations: For the French and Spanish translations of the abstract see Supplementary Materials section