Characterization of a Structural Intermediate of Flavivirus Membrane Fusion

Viral membrane fusion proceeds through a sequence of steps that are driven by triggered conformational changes of viral envelope glycoproteins, so-called fusion proteins. Although high-resolution structural snapshots of viral fusion proteins in their prefusion and postfusion conformations are available, it has been difficult to define intermediate structures of the fusion pathway because of their transient nature. Flaviviruses possess a class II viral fusion protein (E) mediating fusion at acidic pH that is converted from a dimer to a trimer with a hairpin-like structure during the fusion process. Here we show for tick-borne encephalitis virus that exposure of virions to alkaline instead of acidic pH traps the particles in an intermediate conformation in which the E dimers dissociate and interact with target membranes via the fusion peptide without proceeding to the merger of the membranes. Further treatment to low pH, however, leads to fusion, suggesting that these monomers correspond to an as-yet-elusive intermediate required to convert the prefusion dimer into the postfusion trimer. Thus, the use of nonphysiological conditions allows a dissection of the flavivirus fusion process and the identification of two separate steps, in which membrane insertion of multiple copies of E monomers precedes the formation of hairpin-like trimers. This sequence of events provides important new insights for understanding the dynamic process of viral membrane fusion

Capturing a Flavivirus Pre-Fusion Intermediate

During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting ∼60 Å-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements

Reduced naïve CD8+T-cell priming efficacy in elderly adults

International audienceAging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8 + T-cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling na€ ıve CD8 + T-cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8 + T-cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8 + T-cell responses specific for a model antigen. Reduced CD8 + T-cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the na€ ıve CD8 + T-cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging

Water dispersible microbicidal cellulose acetate phthalate film

BACKGROUND: Cellulose acetate phthalate (CAP) has been used for several decades in the pharmaceutical industry for enteric film coating of oral tablets and capsules. Micronized CAP, available commercially as "Aquateric" and containing additional ingredients required for micronization, used for tablet coating from water dispersions, was shown to adsorb and inactivate the human immunodeficiency virus (HIV-1), herpesviruses (HSV) and other sexually transmitted disease (STD) pathogens. Earlier studies indicate that a gel formulation of micronized CAP has a potential as a topical microbicide for prevention of STDs including the acquired immunodeficiency syndrome (AIDS). The objective of endeavors described here was to develop a water dispersible CAP film amenable to inexpensive industrial mass production. METHODS: CAP and hydroxypropyl cellulose (HPC) were dissolved in different organic solvent mixtures, poured into dishes, and the solvents evaporated. Graded quantities of a resulting selected film were mixed for 5 min at 37°C with HIV-1, HSV and other STD pathogens, respectively. Residual infectivity of the treated viruses and bacteria was determined. RESULTS: The prerequisites for producing CAP films which are soft, flexible and dispersible in water, resulting in smooth gels, are combining CAP with HPC (other cellulose derivatives are unsuitable), and casting from organic solvent mixtures containing ≈50 to ≈65% ethanol (EtOH). The films are ≈100 µ thick and have a textured surface with alternating protrusions and depressions revealed by scanning electron microscopy. The films, before complete conversion into a gel, rapidly inactivated HIV-1 and HSV and reduced the infectivity of non-viral STD pathogens >1,000-fold. CONCLUSIONS: Soft pliable CAP-HPC composite films can be generated by casting from organic solvent mixtures containing EtOH. The films rapidly reduce the infectivity of several STD pathogens, including HIV-1. They are converted into gels and thus do not have to be removed following application and use. In addition to their potential as topical microbicides, the films have promise for mucosal delivery of pharmaceuticals other than CAP

Kinder mit Kunstherzunterstützungssystemen im häuslichen Bereich: Ausbildungskonzept und Notfallalgorithmus für Rettungskräfte

Zusammenfassung: Einleitung: Miniaturisierte Herzunterstützungspumpen, sog. Kunstherzsysteme oder "ventricular assist devices" (VADs) bieten die Möglichkeit, diese Systeme im Kindesalter anzuwenden. Durch die lange Wartezeit auf ein geeignetes Spenderorgan sollte bei Kindern, unterstützt mit einem intrakorporealen VAD, die Entlassung nach Hause angestrebt werden. Schwerpunkte vor einem Spitalaustritt sind neben der adäquaten Schulung und Aufklärung des Patienten und deren Familie auch ein Ausbildungs- und Schulungskonzept für die lokalen Rettungskräfte und die Betreuungspersonen vor Ort. Methoden: Es wird ein auf die präklinische Versorgung abgestimmter Notfallalgorithmus für die Erstversorgung von VAD-Patienten vorgestellt sowie das gemeinsam erarbeitete Ausbildungskonzept der lokalen Rettungskräfte und des Kinderspitals Zürich. Schwerpunkte des Schulungsprogramms sind neben der theoretischen Einführung praktische Workshops, "cardiac arrest simulation training" (CAST) sowie die Erstellung eines genau definierten Alarmierungsplans unter Einbezug der lokalen ärztlichen Organisationsstrukturen und der Spezialisten des Kinderspitals. Schlussfolgerung: Die Besonderheiten bei der Versorgung von Kindern am VAD werden vorgestellt und diskutier

A Therapeutic Antibody against West Nile Virus Neutralizes Infection by Blocking Fusion within Endosomes

Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West Nile virus (WNV) that neutralizes infection even after virus has spread to the central nervous system. Herein, we define its mechanism of inhibition. E16 blocks infection primarily at a post-attachment step as antibody-opsonized WNV enters permissive cells but cannot escape from endocytic compartments. These cellular experiments suggest that E16 blocks the acid-catalyzed fusion step that is required for nucleocapsid entry into the cytoplasm. Indeed, E16 directly inhibits fusion of WNV with liposomes. Additionally, low-pH exposure of E16–WNV complexes in the absence of target membranes did not fully inactivate infectious virus, further suggesting that E16 prevents a structural transition required for fusion. Thus, a strongly neutralizing anti–WNV MAb with therapeutic potential is potently inhibitory because it blocks viral fusion and thereby promotes clearance by delivering virus to the lysosome for destruction

Effects of parental acclimation and energy limitation in response to high CO2 exposure in Atlantic cod

Ocean acidification (OA), the dissolution of excess anthropogenic carbon dioxide in ocean waters, is a potential stressor to many marine fish species. Whether species have the potential to acclimate and adapt to changes in the seawater carbonate chemistry is still largely unanswered. Simulation experiments across several generations are challenging for large commercially exploited species because of their long generation times. For Atlantic cod (Gadus morhua), we present first data on the effects of parental acclimation to elevated aquatic CO2 on larval survival, a fundamental parameter determining population recruitment. The parental generation in this study was exposed to either ambient or elevated aquatic CO2 levels simulating end-of-century OA levels (~1100 µatm CO2) for six weeks prior to spawning. Upon fully reciprocal exposure of the F1 generation, we quantified larval survival, combined with two larval feeding regimes in order to investigate the potential effect of energy limitation. We found a significant reduction in larval survival at elevated CO2 that was partly compensated by parental acclimation to the same CO2 exposure. Such compensation was only observed in the treatment with high food availability. This complex 3-way interaction indicates that surplus metabolic resources need to be available to allow a transgenerational alleviation response to ocean acidification

Progressive development of augmentation during long-term treatment with levodopa in restless legs syndrome: results of a prospective multi-center study

The European Restless Legs Syndrome (RLS) Study Group performed the first multi-center, long-term study systematically evaluating RLS augmentation under levodopa treatment. This prospective, open-label 6-month study was conducted in six European countries and included 65 patients (85% treatment naive) with idiopathic RLS. Levodopa was flexibly up-titrated to a maximum dose of 600 mg/day. Presence of augmentation was diagnosed independently by two international experts using established criteria. In addition to the augmentation severity rating scale (ASRS), changes in RLS severity (International RLS severity rating scale (IRLS), clinical global impression (CGI)) were analyzed. Sixty patients provided evaluable data, 35 completed the trial and 25 dropped out. Augmentation occurred in 60% (36/60) of patients, causing 11.7% (7/60) to drop out. Median time to occurrence of augmentation was 71 days. The mean maximum dose of levodopa was 311 mg/day (SD: 105). Patients with augmentation compared to those without were significantly more likely to be on higher doses of levodopa (≥300 mg, 83 vs. 54%, P = 0.03) and to show less improvement of symptom severity (IRLS, P = 0.039). Augmentation was common with levodopa, but could be tolerated by most patients during this 6-month trial. Patients should be followed over longer periods to determine if dropout rates increase with time

Ecological-economic sustainability of the Baltic cod fisheries under ocean warming and acidification

Human-induced climate change such as ocean warming and acidification, threatens marine ecosystems and associated fisheries. In the Western Baltic cod stock socio-ecological links are particularly important, with many relying on cod for their livelihoods. A series of recent experiments revealed that cod populations are negatively affected by climate change, but an ecological-economic assessment of the combined effects, and advice on optimal adaptive management are still missing. For Western Baltic cod, the increase in larval mortality due to ocean acidification has experimentally been quantified. Time-series analysis allows calculating the temperature effect on recruitment. Here, we include both processes in a stock-recruitment relationship, which is part of an ecological-economic optimization model. The goal was to quantify the effects of climate change on the triple bottom line (ecological, economic, social) of the Western Baltic cod fishery. Ocean warming has an overall negative effect on cod recruitment in the Baltic. Optimal management would react by lowering fishing mortality with increasing temperature, to create a buffer against climate change impacts. The negative effects cannot be fully compensated, but even at 3 °C warming above the 2014 level, a reduced but viable fishery would be possible. However, when accounting for combined effects of ocean warming and acidification, even optimal fisheries management cannot adapt to changes beyond a warming of +1.5° above the current level. Our results highlight the need for multi-factorial climate change research, in order to provide the best available, most realistic, and precautionary advice for conservation of exploited species as well as their connected socio-economic systems

Primnoidae (Cnidaria: Octocorallia) of the SW Indian Ocean: new species, genus revisions and systematics

The Indian Ocean is one of the least-studied areas of the world’s largest biome, the deep sea. On an expedition to five seamounts along the SW Indian Ocean Ridge in 2011, thousands of specimens from deep-sea habitats were procured. We propose five new species of Primnoidae, a predominantly deep-sea octocoral family. The new species include three from the genus Narella, and one new species each from Primnoa and Primnoeides; the latter genus is revised and we propose Digitogorgia as its junior synonym. We support the new species placement within Primnoidae through taxonomic descriptions and the most comprehensive molecular phylogenetic analysis of any deep-sea coral family (81 species across 29 genera). We also present a rare example of polar submergence (from the Antarctic shelf into deeper more Northern waters)