Encephalitic alphaviruses exploit caveola-mediated transcytosis at the blood-brain barrier for central nervous system entry

Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB

The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

<p>Abstract</p> <p>Background</p> <p>The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent <it>K</it>_mof the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude.</p> <p>Results</p> <p>We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold.</p> <p>Conclusions</p> <p>Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of <it>K</it>_massociated with kinase enzymes.</p

Colony organization in the green alga Botryococcus braunii (Race B) is specified by a complex extracellular matrix

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form “drapes” between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM

Mycobacterium marinum Escapes from Phagosomes and Is Propelled by Actin-based Motility

Mycobacteria are responsible for a number of human and animal diseases and are classical intracellular pathogens, living inside macrophages rather than as free-living organisms during infection. Numerous intracellular pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsii, exploit the host cytoskeleton by using actin-based motility for cell to cell spread during infection. Here we show that Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of actively inducing actin polymerization within macrophages. M. marinum that polymerized actin were free in the cytoplasm and propelled by actin-based motility into adjacent cells. Immunofluorescence demonstrated the presence of host cytoskeletal proteins, including the Arp2/3 complex and vasodilator-stimulated phosphoprotein, throughout the actin tails. In contrast, Wiskott-Aldrich syndrome protein localized exclusively at the actin-polymerizing pole of M. marinum. These findings show that M. marinum can escape into the cytoplasm of infected macrophages, where it can recruit host cell cytoskeletal factors to induce actin polymerization leading to direct cell to cell spread

Heterologous expression of plasmodial proteins for structural studies and functional annotation

Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the cost-effective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem®. In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed

The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii

When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then “boosted” after 2 days with additional acetate, the cells become “obese” after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of “sensitive” genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes—encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)—are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the “sensitive” genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain

Conserved phosphoryl transfer mechanisms within kinase families and the role of the C8 proton of ATP in the activation of phosphoryl transfer

<p>Abstract</p> <p>Background</p> <p>The kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes.</p> <p>Results</p> <p>A number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH₂of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile.</p> <p>Conclusions</p> <p>The "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety.</p

Global, regional, and national burden of neurological disorders, 1990–2016 : a systematic analysis for the Global Burden of Disease Study 2016

Background: Neurological disorders are increasingly recognised as major causes of death and disability worldwide. The aim of this analysis from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2016 is to provide the most comprehensive and up-to-date estimates of the global, regional, and national burden from neurological disorders. Methods: We estimated prevalence, incidence, deaths, and disability-adjusted life-years (DALYs; the sum of years of life lost [YLLs] and years lived with disability [YLDs]) by age and sex for 15 neurological disorder categories (tetanus, meningitis, encephalitis, stroke, brain and other CNS cancers, traumatic brain injury, spinal cord injury, Alzheimer's disease and other dementias, Parkinson's disease, multiple sclerosis, motor neuron diseases, idiopathic epilepsy, migraine, tension-type headache, and a residual category for other less common neurological disorders) in 195 countries from 1990 to 2016. DisMod-MR 2.1, a Bayesian meta-regression tool, was the main method of estimation of prevalence and incidence, and the Cause of Death Ensemble model (CODEm) was used for mortality estimation. We quantified the contribution of 84 risks and combinations of risk to the disease estimates for the 15 neurological disorder categories using the GBD comparative risk assessment approach. Findings: Globally, in 2016, neurological disorders were the leading cause of DALYs (276 million [95% UI 247–308]) and second leading cause of deaths (9·0 million [8·8–9·4]). The absolute number of deaths and DALYs from all neurological disorders combined increased (deaths by 39% [34–44] and DALYs by 15% [9–21]) whereas their age-standardised rates decreased (deaths by 28% [26–30] and DALYs by 27% [24–31]) between 1990 and 2016. The only neurological disorders that had a decrease in rates and absolute numbers of deaths and DALYs were tetanus, meningitis, and encephalitis. The four largest contributors of neurological DALYs were stroke (42·2% [38·6–46·1]), migraine (16·3% [11·7–20·8]), Alzheimer's and other dementias (10·4% [9·0–12·1]), and meningitis (7·9% [6·6–10·4]). For the combined neurological disorders, age-standardised DALY rates were significantly higher in males than in females (male-to-female ratio 1·12 [1·05–1·20]), but migraine, multiple sclerosis, and tension-type headache were more common and caused more burden in females, with male-to-female ratios of less than 0·7. The 84 risks quantified in GBD explain less than 10% of neurological disorder DALY burdens, except stroke, for which 88·8% (86·5–90·9) of DALYs are attributable to risk factors, and to a lesser extent Alzheimer's disease and other dementias (22·3% [11·8–35·1] of DALYs are risk attributable) and idiopathic epilepsy (14·1% [10·8–17·5] of DALYs are risk attributable). Interpretation: Globally, the burden of neurological disorders, as measured by the absolute number of DALYs, continues to increase. As populations are growing and ageing, and the prevalence of major disabling neurological disorders steeply increases with age, governments will face increasing demand for treatment, rehabilitation, and support services for neurological disorders. The scarcity of established modifiable risks for most of the neurological burden demonstrates that new knowledge is required to develop effective prevention and treatment strategies. Funding: Bill & Melinda Gates Foundation

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure