The organization and number of microtubules (MTs) in a cell depend on the proper regulation of MT nucleation. Currently, the mechanism of nucleation is the most poorly understood aspect of MT ...dynamics. XMAP215/chTOG/Alp14/Stu2 proteins are MT polymerases that stimulate MT polymerization at MT plus ends by binding and releasing tubulin dimers. Although these proteins also localize to MT organizing centers and have nucleating activity in vitro, it is not yet clear whether these proteins participate in MT nucleation in vivo. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, the XMAP215 ortholog Alp14 is critical for efficient MT nucleation in vivo. In multiple assays, loss of Alp14 function led to reduced nucleation rate and numbers of interphase MT bundles. Conversely, activation of Alp14 led to increased nucleation frequency. Alp14 associated with Mto1 and γ-tubulin complex components, and artificially targeting Alp14 to the γ-tubulin ring complexes (γ-TuRCs) stimulated nucleation. In imaging individual nucleation events, we found that Alp14 transiently associated with a γ-tubulin particle shortly before the appearance of a new MT. The transforming acidic coiled-coil (TACC) ortholog Alp7 mediated the localization of Alp14 at nucleation sites but not plus ends, and was required for efficient nucleation but not for MT polymerization. Our findings provide the strongest evidence to date that Alp14 serves as a critical MT nucleation factor in vivo. We suggest a model in which Alp14 associates with the γ-tubulin complex in an Alp7-dependent manner to facilitate the assembly or stabilization of the nascent MT.
•Alp14 (XMAP215) and Alp7 (TACC) promote microtubule nucleation in vivo•Alp14 interaction with Mto1 and the γ-tubulin complex is dependent on Alp7•Increasing the affinity of Alp14 for Mto1 increases nucleation frequency•Timed appearance of Alp14 at the nucleation site suggests a nucleation timeline
Microtubules are dynamic polymers that help to organize cellular contents and divide the cell. New microtubules arise by a process of nucleation, in which tubulin subunits are stitched together to begin forming a hollow tube. Flor-Parra et al. identify XMAP215/Alp14 as a nucleation factor that facilitates the assembly of the microtubule.
West Nile virus (WNV) is a neurovirulent mosquito-borne flavivirus, which main natural hosts are birds but it also infects equines and humans, among other mammals. As in the case of other ...plus-stranded RNA viruses, WNV replication is associated to intracellular membrane rearrangements. Based on results obtained with a variety of viruses, different cellular processes have been shown to play important roles on these membrane rearrangements for efficient viral replication. As these processes are related to lipid metabolism, fatty acid synthesis, as well as generation of a specific lipid microenvironment enriched in phosphatidylinositol-4-phosphate (PI4P), has been associated to it in other viral models. In this study, intracellular membrane rearrangements following infection with a highly neurovirulent strain of WNV were addressed by means of electron and confocal microscopy. Infection of WNV, and specifically viral RNA replication, were dependent on fatty acid synthesis, as revealed by the inhibitory effect of cerulenin and C75, two pharmacological inhibitors of fatty acid synthase, a key enzyme of this process. However, WNV infection did not induce redistribution of PI4P lipids, and PI4P did not localize at viral replication complex. Even more, WNV multiplication was not inhibited by the use of the phosphatidylinositol-4-kinase inhibitor PIK93, while infection by the enterovirus Coxsackievirus B5 was reduced. Similar features were found when infection by other flavivirus, the Usutu virus (USUV), was analyzed. These features of WNV replication could help to design specific antiviral approaches against WNV and other related flaviviruses.
Abstract
Background
Viral rewiring of host bioenergetics and immunometabolism may provide novel targets for therapeutic interventions against viral infections. Here, we have explored the effect on ...bioenergetics during the infection with the mosquito-borne flavivirus West Nile virus (WNV), a medically relevant neurotropic pathogen causing outbreaks of meningitis and encephalitis worldwide.
Results
A systematic literature search and meta-analysis pointed to a misbalance of glucose homeostasis in the central nervous system of WNV patients. Real-time bioenergetic analyses confirmed upregulation of aerobic glycolysis and a reduction of mitochondrial oxidative phosphorylation during viral replication in cultured cells. Transcriptomics analyses in neural tissues from experimentally infected mice unveiled a glycolytic shift including the upregulation of hexokinases 2 and 3 (
Hk2
and
Hk3
) and pyruvate dehydrogenase kinase 4 (
Pdk4
). Treatment of infected mice with the Hk inhibitor, 2-deoxy-D-glucose, or the Pdk4 inhibitor, dichloroacetate, alleviated WNV-induced neuroinflammation.
Conclusions
These results highlight the importance of host energetic metabolism and specifically glycolysis in WNV infection in vivo. This study provides proof of concept for the druggability of the glycolytic pathway for the future development of therapies to combat WNV pathology.
Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that affects humans and can cause severe neurological complications, including Guillain-Barré syndrome and microcephaly. Since 2007 there ...have been three large outbreaks; the last and larger spread in the Americas in 2015. Actually, ZIKV is circulating in the Americas, Southeast Asia, and the Pacific Islands, and represents a potential pandemic threat. Given the rapid ZIKV dissemination and the severe neurological and teratogenic sequelae associated with ZIKV infection, the development of a safe and efficacious vaccine is critical. In this study, we have developed and characterized the immunogenicity and efficacy of a novel ZIKV vaccine based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the ZIKV prM and E structural genes (termed MVA-ZIKV). MVA-ZIKV expressed efficiently the ZIKV structural proteins, assembled in virus-like particles (VLPs) and was genetically stable upon nine passages in cell culture. Immunization of mice with MVA-ZIKV elicited antibodies that were able to neutralize ZIKV and induced potent and polyfunctional ZIKV-specific CD8
T cell responses that were mainly of an effector memory phenotype. Moreover, a single dose of MVA-ZIKV reduced significantly the viremia in susceptible immunocompromised mice challenged with live ZIKV. These findings support the use of MVA-ZIKV as a potential vaccine against ZIKV.
Zika virus (ZIKV), a mosquito-borne flavivirus, was an almost neglected pathogen until its introduction in the Americas in 2015, where it has been responsible for a threat to global health, causing a ...great social and sanitary alarm due to its increased virulence, rapid spread, and an association with severe neurological and ophthalmological complications. Currently, no specific antiviral therapy against ZIKV is available, and treatments are palliative and mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration. Nevertheless, lately, search for antivirals has been a major aim in ZIKV investigations. To do so, screening of libraries from different sources, testing of natural compounds, and repurposing of drugs with known antiviral activity have allowed the identification of several antiviral candidates directed to both viral (structural proteins and enzymes) and cellular elements. Here, we present an updated review of current knowledge about anti-ZIKV strategies, focusing on host-directed antivirals as a realistic alternative to combat ZIKV infection.
Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve ...transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress.
We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20
during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20
N-terminus through a canonical MAPK docking site. Most important, the Cdc20
pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit.
Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20
turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.
The Flavivirus is a genus of RNA viruses that includes multiple long known human, animal, and zoonotic pathogens such as Dengue virus, yellow fever virus, West Nile virus, or Japanese encephalitis ...virus, as well as other less known viruses that represent potential threats for human and animal health such as Usutu or Zika viruses. Flavivirus replication is based on endoplasmic reticulum-derived structures. Membrane remodeling and accumulation of viral factors induce endoplasmic reticulum stress that results in activation of a cellular signaling response termed unfolded protein response (UPR), which can be modulated by the viruses for their own benefit. Concomitant with the activation of the UPR, an upregulation of the autophagic pathway in cells infected with different flaviviruses has also been described. This review addresses the current knowledge of the relationship between endoplasmic reticulum stress, UPR, and autophagy in flavivirus-infected cells and the growing evidences for an involvement of these cellular pathways in the replication and pathogenesis of these viruses.
Nitrate-rich beetroot juice supplementation has been extensively used to increase exercise economy in different populations. However, its use in elite distance runners, and its potential effects on ...biomechanical aspects of running have not been properly investigated. This study aims to analyze the potential effects of 15 days of beetroot juice supplementation on physiological, psychological and biomechanical variables in elite runners.
Twelve elite middle and long-distance runners (age = 26.3 ± 5.1yrs, VO2Max = 71.8±5.2 ml*kg-1*min-1) completed an incremental running test to exhaustion on a treadmill before and after a 15-days supplementation period, in which half of the group (EG) consumed a daily nitrate-rich beetroot juice and the other group (PG) consumed a placebo drink. Time to exhaustion (TEx), running economy, vastus lateralis oxygen saturation (SmO2), leg stiffness and rate of perceived exertion (RPE) were measured at 15, 17.1 and 20 km/h during the incremental test.
Likely to very likely improvements in EG were observed for the RPE (Standardized mean difference (SMD) = -2.17, 90%CI = -3.23, -1.1), SmO2 (SMD = 0.72, 90%CI = 0.03, 1.41) and TEx (SMD = 1.18, 90%CI = -0.14, 2.5) in comparison with PG. No other physiological or biomechanical variable showed substantial improvements after the supplementation period.
Fifteen days of nitrate-rich beetroot juice supplementation produced substantial improvements in the time to exhaustion in elite runners; however, it didn't produce meaningful improvements in running economy, VO2Max or mechanical parameters.
To identify and quantify associations between baseline characteristics on hospital admission and mortality in patients with COVID-19 at a tertiary hospital in Spain.
This retrospective case series ...included 238 patients hospitalized for COVID-19 at Hospital Universitario Clínico San Cecilio (Granada, Spain) who were discharged or who died. Electronic medical records were reviewed to obtain information on sex, age, personal antecedents, clinical features, findings on physical examination, and laboratory results for each patient. Associations between mortality and baseline characteristics were estimated as hazard ratios (HR) calculated with Cox regression models. Series mortality was 25.6%. Among patients with dependence for basic activities of daily living, 78.7% died, and among patients residing in retirement homes, 80.8% died. The variables most clearly associated with a greater hazard of death were age (3% HR increase per 1-year increase in age; 95%CI 1-6), diabetes mellitus (HR 2.42, 95%CI 1.43-4.09), SatO2/FiO2 ratio (43% HR reduction per 1-point increase; 95%CI 23-57), SOFA score (19% HR increase per 1-point increase, 95%CI 5-34) and CURB-65 score (76% HR increase per 1-point increase, 95%CI 23-143).
The patients residing in retirement homes showed great vulnerability. The main baseline factors that were independently associated with mortality in patients hospitalized for COVID-19 were older age, diabetes mellitus, low SatO2/FiO2 ratio, and high SOFA and CURB-65 scores.
West Nile virus (WNV) is a neurovirulent mosquito-borne flavivirus. High WNV virulence was mainly associated with lineage 1 strains, but recent outbreaks have unveiled circulation of highly virulent ...lineage 2 strains. Co-expression of flavivirus prM and E glycoproteins drives the assembly of recombinant subviral particles (RSPs) that share antigenic features with virions. Mouse immunization with lineage 1 WNV RSPs induced a potent humoral response against WNV with production of neutralizing antibodies. A single inoculation of RSPs formulated with Al(OH)3 as adjuvant protected mice against a lethal challenge with WNV strains from lineage 1 or 2. The cross-reactivity of the response elicited by these RSPs was analyzed against the related flavivirus Usutu virus (USUV), which shares multiple ecological and antigenic features with WNV. Immunization with WNV-RSPs increased specific, although low, antibody titers found upon subsequent USUV infection.
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